Moraxella catarrhalis outer membrane protein-106 polypeptide, gene sequence and uses thereof

ABSTRACT

The invention discloses the  Moraxella catarrhalis  outer membrane protein-106 (OMP106) polypeptide, polypeptides derived therefrom (OMP106-derived polypeptides), nucleotide sequences encoding OMP106 polypeptides, and antibodies that specifically bind the OMP106 polypeptide and/or OMP106-derived polypeptides. Also disclosed are immunogenic, prophylactic or therapeutic compositions, including vaccines, comprising OMP106 polypeptide and/or OMP 106-derived polypeptides. The invention additionally discloses methods of inducing immune responses to  M. catarrhalis  and  M. catarrhalis  OMP 106 polypeptides and OMP 106-derived polypeptides in animals.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a division of application Ser. No. 11/580,854, filedOct. 16, 2006, now U.S. Pat. No. 7,576,179, which is a division ofApplication. No. 09/813,214 filed Mar. 20, 2001, which is a division ofApplication. No. 08/968,685, filed Nov. 12, 1997, now U.S. Pat. No.6,214,981, which is a continuation-in-part of Application. No.08/642,712, filed May 3, 1996, now U.S. Pat. No. 7,341,727, thedisclosures of which are all incorporated herein by reference in theirentirety.

INTRODUCTION

The present invention generally relates to the outer membraneprotein-106 (OMP106) polypeptide of Moraxella catarrhalis. The inventionencompasses a purified OMP106 polypeptide and polypeptides derivedtherefrom (OMP106-derived polypeptides). The invention also encompassesantibodies, including cytotoxic antibodies, that specifically bind theOMP106 polypeptide and/or OMP106-derived polypeptides. The inventionfurther encompasses prophylactic or therapeutic compositions, includingvaccines, that comprise OMP106 polypeptide and/or OMP106-derivedpolypeptides. The invention additionally provides methods of inducingimmune responses to M. catarrhalis in mammals. The invention furtherprovides isolated nucleotide sequences encoding the OMP106 polypeptideand OMP106-derived polypeptides, vectors having said sequences, and hostcells containing said vectors.

BACKGROUND OF THE INVENTION

Moraxella catarrhalis, also known as Moraxella (Branhamella) catarrhalisor Branhamella catarrhalis and formerly known as Neisseria catarrhalisor Micrococcus catarrhalis, is a gram-negative bacterium frequentlyfound in the respiratory tract of humans. M. catarrhalis, originallythought to be a harmless commensal organism, is now recognized as animportant pathogen in upper and lower respiratory tract infections inanimals. In humans, M. catarrhalis causes serious lower respiratorytract infections in adults with chronic lung disease, systemicinfections in immunocompromised patients, and otitis media and sinusitisin infants and children. See Helminen et al., 1993, Infect. Immun.61:2003-2010; Catlin, B. W., 1990, Clin. Microbiol. Rev. 3:293 320; andreferences cited therein.

2.1. Outer Membrane Proteins and Protective Antibodies

The outer surface components of Moraxella catarrhalis have been studiedin attempts to understand the pathogenic process of M. catarrhalisinfections and to develop useful therapeutic treatments and prophylacticmeasures against such infections. The outer membrane proteins (OMPs) inparticular have received considerable attention as possible virulencefactors and as potential vaccine antigens. M. catarrhalis has about 10to 20 different OMPs with 6 to 8 of these, OMPs A to H, as thepredominate species (Murphy and Loeb, 1989, Microbial Pathogen.6:159-174). The molecular weights of OMPs A to H range from 97 to 20 kD,respectively. See Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765;Helminen et al., 1993, Infect. Immun. 61:2003-2010; Murphy et al, 1993,Molecul. Microbiol. 10: 87-97; and Sarwar et al, 1992, Infect. Immun.60:804 809. Comparisons of protein profiles by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of outer membranepreparations from 50 M. catarrhalis strains show nearly homogeneouspatterns of OMPs A to H (Bartos and Murphy, 1988, J. Infect. Dis.158:761 765).

In addition to OMPs A to H, a high molecular weight OMP, designatedHMW-OMP, having an apparent mass of 350 to 720 kD by SDS-PAGE has alsobeen identified as another prominent surface component present in manystrains of M. catarrhalis. HMW-OMP upon formic acid denaturationproduces a single band of 120 to 140 kD and, thus, appears to be anoligomeric protein (Klingman and Murphy, 1994, Infect. Immun. 62:11501155). HMW-OMP appears to be the same protein as that designated UspA byHelminen et al., (1994, J. Infect. Dis. 170:867-872) and shown to bepresent in a number of M. catarrhalis strains.

In intact bacterium or bacterially-derived outer membrane vesicles,several of the above-identified OMPs present surface-exposed epitopesthat elicit the production of antibodies that bind the OMPs. Theseantigenic OMPs include OMP E and OMP G (Murphy and Bartos, 1989, Infect.Immun. 57:2938-2941); OMP C/D (Sarwar et al., 1992, Infect. Immun.60:804-809); CopB, an 80 kD OMP, (Helminen et al., 1993, Infect. Immun.61:2003 2010); and UspA (Helminen et al., 1994, J. Infect. Dis. 170:867872).

The therapeutic potential of antibodies to surfaced-exposed epitopes ofCopB and UspA has been evaluated in an animal model. The model involveddirect bolus inoculation of lungs of BALB/c VAF/Plus mice with acontrolled number of M. catarrhalis cells and subsequent examination ofthe rate of pulmonary clearance of the bacteria (Unhanand et al., 1992,J. Infect. Dis. 165:644-650). Different clinical isolates of the M.catarrhalis exhibited different rates of clearance that correlated withthe level of granulocyte recruitment into the infection site. Passiveimmunization with a monoclonal antibody directed to a surface-exposedepitope of either CopB or UspA increased the rate of pulmonary clearanceof M. catarrhalis (Helminen et al., 1993, Infect. Immun. 61:2003-2010;Helminen et al., 1994, J. Infect. Dis. 170:867-872).

2.2. Bacterial/Host Cell Adherence and Hemagglutination

The adherence of bacterial pathogens to a host cell surface promotescolonization and initiates pathogenesis. See, E. H. Beachey, 1981, J.Infect. Dis. 143:325-345. Gram-negative bacteria typically expresssurface lectins that bind to specific oligosaccharides of glycoproteinsand/or glycolipids on the host cell surface. Such lectins are oftenassociated with pili or fimbriae. Bacterial adherence can also occur bynon-specific binding resulting from hydrophobic and/or chargeinteraction with the host cell surface.

The mechanism of M. catarrhalis adherence to cells of the respiratorytract remains poorly understood. The organism adheres to cultured humanoropharyngeal epithelial cells (Mbaki et al., 1987, Tohuku J. Exp. Med.153:111 121). A study by Rikitomi et al. suggests that fimbriae may havea role in the adherence to such cells as fimbriae denaturation ortreatment with anti-fimbriae antibodies reduced adherence by fimbriatedstrains (Rikitomi et al., 1991, Scand. J. Infect. Dis. 23:559-567).Fimbriae mediated binding, however, cannot be the sole basis of thisadherence as the most highly adhering strain, among the severalexamined, was a non-fimbriated strain.

Hemagglutination reactions often replace the more complicated adherenceassays in classifying bacterial adhesins. However, Rikitomi et al. foundno correlation between human oropharyngeal epithelial cell adherence andhemagglutination by M. catarrhalis strains (Id.). That is three highlyadhering strains did not agglutinate human erythrocytes. Thus, differentbinding mechanisms are involved in human oropharyngeal epithelial celladherence and hemagglutination.

By contrast, a recent study by Kellens et al. suggests thathemagglutination by M. catarrhalis is correlated with host celladherence (Kellens et al., 1995, Infection 23:37-41). However, thisstudy employed an adherence assay based on bacterial binding to porcinetracheal sections. It is unclear whether porcine tracheal tissue can beconsidered homologous to human respiratory tract tissue with respect toadherence by pathogenic strains of M. catarrhalis.

Notwithstanding the problematic adherence assay, Kellens et al. examinedthe hemagglutination activities of eighty-some clinical isolates of M.catarrhalis (Kellens et al., 1995, Infection 23:37-41). Nearlythree-quarters of the examined strains agglutinated human, rabbit,guinea pig, dog or rat erythrocytes, while the remaining strains didnot. The agglutination activities for some of the hemagglutinatingstains were further characterized and shown to be calcium ion dependentand inhibited by trypsin digestion or high-temperature treatment oraddition of D-glucosamine or D-galactosamine.

A survey of hemagglutinating and non-hemagglutinating M. catarrhalisstrains by Tucker et al. has shown that all strains bind the glycolipidgangliotetraosylceramide but only hemagglutinating strains bind theglycolipid globotetraosylceramide (Tucker et al., 1994, Annual Meetingof Amer. Soc. Microbiol., Abstract No. D124). Moreover, M. catarrhalishemagglutination activity was shown to be inhibited by variousmonosaccharides that comprise the carbohydrate moiety ofglobotetraosylceramide. These observations led Tucker et al. to suggestthat M. catarrhalis hemagglutinates by binding toglobotetraosylceramides in the cell membranes of susceptibleerythrocytes, including those of human red blood cells. To date, noprior art has identified a molecule on the outer surface of M.catarrhalis that is responsible for either host cell adherence orhemagglutination.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an indication thatsuch reference is available as prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention encompasses the OMP106 polypeptide of M.catarrhalis and OMP106-derived polypeptides and methods for making saidpolypeptides. The invention also encompasses antisera and antibodies,including cytotoxic antibodies, specific for the OMP106 polypeptideand/or OMP106-derived polypeptides. The invention further encompassesimmunogenic, prophylactic or therapeutic compositions, includingvaccines, comprising one or more of said polypeptides. The inventionadditionally encompasses nucleotide sequences encoding saidpolypeptides. The invention further encompasses immunogenic,prophylactic or therapeutic compositions, including vaccines, comprisingan attenuated or inactivated non-hemagglutinating M. catarrhaliscultivar.

The present invention has many utilities. For example, the OMP106polypeptide and OMP106-derived polypeptides may be used as ligands todetect antibodies elicited in response to M. catarrhalis infections(e.g., in diagnosing M. catarrhalis infections). The OMP106 polypeptideand OMP106-derived polypeptides may also be used as immunogens forinducing M. catarrhalis-specific antibodies. Such antibodies are usefulin immunoassays to detect M. catarrhalis in biological specimens. Thecytotoxic antibodies of the invention are useful in passiveimmunizations against M. catarrhalis infections. The OMP106 polypeptideand OMP106-derived polypeptides may further be used as activeingredients in vaccines against M. catarrhalis infections.

The invention is based on the surprising discovery that hemagglutinatingM. catarrhalis strains and cultivars have an outer membrane protein,OMP106 polypeptide, which is about 180 kD to about 230 kD in molecularweight, and that non-hemagglutinating M. catarrhalis strains andcultivars either do not have OMP106 polypeptide or haveinappropriately-modified OMP106 polypeptide which is inactive inhemagglutination and not silver-stainable. The invention is furtherbased on the discovery that polyclonal antiserum induced by OMP106polypeptide isolated from a hemagglutinating M. catarrhalis strain hascytotoxic activity against a different hemagglutinating M. catarrhalisstrain but not against a non-hemagglutinating M. catarrhalis strain.

3.1. DEFINITIONS AND ABBREVIATIONS

anti-OMP106=anti-OMP106 polypeptide antibody or antiserum

ATCC®=American Type Culture Collection

blebs=naturally occurring outer membrane vesicles of M. catarrhalis

Gb₄=GalNAcβ1-3Galα1-4Galβ1-4Glc1-1 Ceramide

HA=hemagglutinatingimmuno-reactive=capable of provoking a cellular or humoral immuneresponsekD=kilodaltonsM. catarrhalis=Mc; Moraxella catarrhalis; Moraxella (Branhamella)catarrhalis; Branhamella catarrhalis; Neisseria catarrhalis; orMicrococcus catarrhalisNHA=non-hemagglutinatingOG=n-octyl β-D-glucopyranoside or octyl glucosideOMP106=the outer membrane protein-106 polypeptide of Moraxellacatarrhalis, having a molecular weight of about 180 kD to 230 kD bySDS-PAGE; extractable from blebs or intact cells of M. catarrhalis by OGor sarkosyl detergentOMP106-derived=fragment of the OMP106 polypeptide; polypeptide variantof wild-type OMP106 polypeptide or fragment thereof, containing one ormore amino acid deletions, insertions or substitutions; or chimericprotein comprising a heterologous polypeptide fused to the C-terminal orN-terminal or internal segment of a whole or a portion of the OMP106polypeptideOMP=outer membrane proteinOMPs=outer membrane proteinsPBS=phosphate buffered salinePAG=polyacrylamide gelpolypeptide=a peptide of any length, preferably one having ten or moreamino acid residuesSDS=sodium dodecylsulfateSDS-PAGE=sodium dodecylsulfate polyacrylamide gel electrophoresis

Nucleotide or nucleic acid sequences defined herein are represented byone-letter symbols for the bases as follows:

A (adenine)C (cytosine)G (guanine)T (thymine)U (uracil)

M (A or C) R (A or G) W (A or T/U) S(C or G) Y (C or T/U) K (G or T/U) V(A or C or G; not T/U) H (A or C or T/U; not G) D (A or G or T/U; not C)B (C or G or T/U; not A)

N (A or C or G or T/U) or (unknown)

Peptide and polypeptide sequences defined herein are represented byone-letter symbols for amino acid residues as follows:

A (alanine)R (arginine)N (asparagine)D (aspartic acid)C (cysteine)Q (glutamine)E (glutamic acid)G (glycine)H (histidine)I (isoleucine)L (leucine)K (lysine)M (methionine)F (phenylalanine)P (proline)S (serine)T (threonine)W (tryptophan)Y (tyrosine)V (valine)X (unknown)

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, non-limiting examplesof specific embodiments of the invention and the appended figures.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1: Denaturing PAGE comparison of outer membrane protein profiles ofM. catarrhalis blebs or octyl glucoside (OG) extracts of whole M.catarrhalis cells. The numbers over the lanes refer to the ATCC®(American Type Culture Collection) strain designations. A prestainedSDS-PAGE standard (BioRad catalog #161-0305) was used as molecularweight markers. The standard consisted of the following polypeptideswith their approximate molecular weights noted in parenthesis: rabbitmuscle phosphorylase B (106 kD); bovine serum albumin (80 kD); hen eggwhite ovalbumin (49.5 kD); bovine carbonic anhydrase (32.5 kD); soybeantrypsin inhibitor (27.5 kD); hen egg white lysozyme (18.5 kD). Thepositions of the molecular weight markers in the gel are noted on theleft side of the drawing by arrows with the molecular weights (kD) ofsome of the markers above the arrows.

FIG. 2: Results from overlaying thin layer chromatograms of glycolipidswith ¹²⁵I-labeled outer membrane blebs. In Panels A C, Lane 1 containsglycolipid standards indicated on the left; Lane 2 contains asialo-GM₁;Lane 3 contains Gb₃, Gb₄, and Forssman antigen; and Lane 4 contains aFolch extraction of human erythrocytes. The chromatogram shown in PanelA is stained with orcinol, the chromatogram shown in Panel B isoverlayed with ¹²⁵I-labeled blebs of ATCC® (American Type CultureCollection) strain 8176 (a non-hemagglutinating strain), and thechromatogram shown in Panel C is overlayed with ¹²⁵I-labeled blebs ofATCC® (American Type Culture Collection) strain 49143 (ahemagglutinating strain). Only the hemagglutinating strain bound to theGb₄ glycolipid band in the third and fourth lanes.

FIG. 3: Protein profiles by silver staining of octyl glucoside extractsof outer membrane proteins following digestion of the M. catarrhaliscells with the proteases indicated in the figure. The hemagglutinationactivity of the cells following the digestion is indicated below thefigure in the row labeled HA. The molecular weight markers used are asper FIG. 1.

FIG. 4: Comparison of protein profiles by silver staining of outermembrane proteins from various ATCC® (American Type Culture Collection)strains of M. catarrhalis. The strain designations are indicated abovethe lanes. The hemagglutination activity of the strains are indicated inthe row labeled HA below the figure. Note a protein having an apparentmolecular weight greater than that of rabbit muscle phosphorylase B (106kD) is common to the hemagglutinating strains, but is absent in thenon-hemagglutinating strains. This polypeptide is designated OMP106. Themolecular weight markers used are as per FIG. 1.

FIG. 5: Comparison of protein profiles by silver staining of outermembrane proteins from two M. catarrhalis ATCC® (American Type CultureCollection) 49143 cultivars: 49143 (hemagglutinating cultivar) and49143-NHA (non-hemagglutinating cultivar). The hemagglutinationactivities of the cultivars are indicated below the figure in the rowlabeled HA. Note the absence of the OMP106 polypeptide band (indicatedby <) in the non-hemagglutinating cultivar. The molecular weight markersused are as per FIG. 1.

FIG. 6: Molecular weight estimation of OMP106 in a 6% denaturingpolyacrylamide gel using OG extracts of ATCC® (American Type CultureCollection) strain 49143 that were incubated in sample buffer at either25° C. or 100° C. prior to application to the gel. Proteins in the gelwere visualized by reductive silver staining. Note that the OMP106polypeptide band (indicated by the <) is seen only in the sampleincubated at 100° C. A broad range SDS-PAGE standard (BioRad catalog#161-0317) was used as molecular weight markers. The standard consistedof the following polypeptides (approximate molecular weights noted inparenthesis): rabbit skeletal muscle myosin (200 kD); E. coliβ-galactosidase (116 kD); rabbit muscle phosphorylase B (97.4 kD);bovine serum albumin (66.2 kD). The positions of the molecular weightmarkers in the gel are noted on the right side of the figure by arrowswith the molecular weights (kD) of the markers above the arrows.

FIG. 7: Southern blot analysis of DraI and HindIII restrictionendonuclease digests of M. catarrhalis chromosomal DNA probed withMc5-72. DNA of M. catarrhalis strain 49143 was digested with DraI orHindIII. Southern analysis of the digested DNA was carried out usingMc5-72 (SEQ ID NO:4) as the probe. The high stringency wash was 2×SSC,1% SDS at 50° C. for about 20 to about 30 minutes. Lane 1 containsHindIII digest; the hybridizing band has an approximate size of 8.0 kb.Lane 2 contains DraI digest: the hybridizing band has an approximatesize of 4.2 kb.

FIGS. 8A and 8B: Western Blots of protein extracts of M. catarrhalis andrelated species using a rabbit antiserum to OMP106 as the probe (FIG.8A), compared to the reactivity of the serum prior to immunization ofthe rabbit with OMP106 (FIG. 8B). Samples in the lanes of FIGS. 8A and8B are as follows: Lane A, M. catarrhalis; Lane B, Moraxella ovis; LaneC, Moraxella lacunata; Lane D, Moraxella osloensis; Lane E, Moraxellabovis; Lane F, Neisseria meningitidis; Lane G, Neisseria gonorrhoeae.The molecular weight markers used are as per FIG. 1.

FIG. 9A: Western blot demonstrating that a rabbit antiserum to theOMP106 polypeptide from M. catarrhalis ATCC® (American Type CultureCollection) 49143 cross-reacts with a polypeptide of a similar molecularweight in a number of HA and NHA strains of M. catarrhalis (the locationof the OMP106 polypeptide is indicated by the arrow). The Westernexamined octyl glucoside extracts of various M. catarrhalis strains. TheATCC® (American Type Culture Collection) accession numbers of thestrains are indicated at the top of the lanes. The transfer and Westernblot procedures used were identical to those used to obtain the blotsshown in FIG. 8.

FIG. 9B: Western blot of the same extracts as those in FIG. 9A using thepre-immune serum corresponding to that used in FIG. 9A.

FIG. 10: Western blot using a monoclonal antibody to OMP106 polypeptideSRB #1 to probe proteins resolved on a 4 to 20% SDS-polyacrylamide gel.OMP106 must be heated to 100° C. before it will resolve at 190 kD on agel. The samples are as follows: (1) molecular weight standards; (2)detergent extract containing outer membrane proteins of M. catarrhalisthat were heated to 100° C.; (3) purified OMP106 heated to 100° C.; (4)detergent extract containing outer membrane proteins of M. catarrhalisthat were incubated at room temperature; and (5) purified OMP106incubated at room temperature.

FIG. 11: organization of the OMP106 locus and fragments that weresubcloned. 72 refers to the approximate location of the 72 bp DNAfragment. Plasmid p omp N/P refers to a omp106 DNA fragment obtained bydigestion with NotI/PstI and subcloned into pBluescriptII SK. AClaI/PstI restriction fragment was obtained from p omp N/P and subclonedinto pBluescriptII SK to yield the plasmid p omp C/P. A PCR producthaving an approximate size of 3.5 kb was generated using genomic DNA orphageλ omp106.6 DNA as a template, digested with PstI and EcoRI andcloned into PstI/EcoRI digested pBluescriptII SK to yield the plasmid pomp P/R. Physical mapping of the pBK omp R/H located a unique XbaI siteapproximately 1.5 kb upstream from the HindIII site. Sequences betweenthis XbaI site and XbaI in the polylinker were deleted and therecircularized phagemid was designated as pBK omp R/X. Plasmid p omp106X containing the entire open reading frame of OMP106 was constructedfrom pBK omp R/X, p omp C/P, and p omp P/R; see Section 9 for details.

FIGS. 12A and 12B: Map of OMP106 and OMP106 deletion mutants. Theorganization of the omp 106 locus in the wild-type strain (FIG. 12A)compared to the structure imposed on the locus after the gene-targetingconstruct has been inserted by homologous recombination (FIG. 12B) isshown. Probe 1 and probe 2 designate DNA fragments used for Southernanalysis. 72 refers to the approximate location of the 72 bp fragmentdescribed in the text. Thin lines under the construct indicate thelength of DNA fragments generated by cutting DNA from wild-type andknockout strains with ClaI or ClaI/EcoRI.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Hemagglutinating andNon-Hemagglutinating Cultivars

The invention provides an isolated or a substantially pure OMP106polypeptide of M. catarrhalis. The OMP106 polypeptide comprises thewhole or a subunit of a protein embedded in or located on the outersurface of the outer membrane of hemagglutinating (HA) strains and manynonhemagglutinating (NHA) strains and cultivars of M. catarrhalis.OMP106 contributes directly or indirectly to the hemagglutinationphenotype of the HA strains and cultivars. According to the invention,HA M. catarrhalis cells agglutinate human or rabbit erythrocytes in anystandard hemagglutination assay, such as the one taught bySoto-Hernandez et al. 1989, J. Clin. Microbiol. 27:903-908. Although notintending to be limited to any particular mechanism of action, it ispresently envisaged that M. catarrhalis agglutinates erythrocytes bybinding to the globotetrose (Gb₄) moiety of glycolipid and glycoproteinreceptors on the host cell surfaces and that the hemagglutinationactivity is mediated in part by appropriately modified OMP106polypeptide, which has the particular property of being susceptible tosilver staining. By contrast, unmodified or inappropriately modifiedOMP106 polypeptide is neither active in mediating hemagglutination norsilver-stainable. Moreover, OMP106 polypeptide is the only polypeptidehaving an apparent molecular weight of about 180 kD to about 230 kD inSDS-PAGE that is OG- or sarkosyl-extractable from HA or NHA M.catarrhalis blebs or intact cells.

The hemagglutination activity of HA M. catarrhalis cells is inhibited byglobotetrose (GalNAcβ1-3 Galα1-4Galβ1-4Glcβ1; Gb₄) and themonosaccharides that comprise Gb4, including N-acetyl-D-galactosamine,D-galactose and glucose, and derivatives thereof, such asmethyl-α-galactose or methyl-α-galactose. The hemagglutination activityof HA M. catarrhalis cells is also inhibited by relatively higherconcentrations of a number of other sugars including but not limited toD-mannose, L-fucose, D-glucose, and N-acetyl-D-glucosamine.

The hemagglutination activity and the OMP106 polypeptide of intact HA M.catarrhalis cells are both reduced or destroyed by digestion of intactM. catarrhalis cells by various proteases including, but not limited to,LCK (Nα-ptosyl-L-lysine chloro methyl ketone [also known as1-chloro-3-tosylamino-7-amino-L-2-heptanone])-treated chymotrypsin,proteinase K and TPCK (N-tosyl-L-phenylalanine chloromethylketone)-treated trypsin. Protease V8 digestion of intact HA M.catarrhalis cells, however, affects neither the hemagglutinationactivity nor the physical integrity of the OMP106 polypeptide of suchcells.

A non-hemagglutinating (NHA) cultivar may be derived from a HA M.catarrhalis strain or cultivar by serial passage in static liquidcultures (i.e., liquid cultures maintained at 35° C. without shaking).For example, a HA M. catarrhalis strain or cultivar is grown in MuellerHinton broth and every five days an inoculum is taken from the surfaceof the static culture to inoculate a subsequent static culture. Thepreferred inoculum is any floating mat of cells at the surface of theculture. Passaging in static cultures is maintained until a NHA cultivaris produced. A NHA cultivar of the invention may be used to produceprotective vaccines, such as whole cell vaccines, against M. catarrhalisinfections.

By contrast, the hemagglutinating phenotype of a HA M. catarrhalisstrain or cultivar can be maintained by passaging the strain or cultivarin shaking liquid cultures. In an embodiment, a HA M. catarrhalis strainor cultivar is grown in Mueller Hinton broth at 35 to 37° C. withshaking at about 200 RPM and passaged every 24 to 48 hours. Thehemagglutinating phenotype of a HA M. catarrhalis strain or cultivaralso can be maintained by passaging on solid media. For example, a HA M.catarrhalis strain or cultivar is grown on a plate containing blood agaror Mueller Hinton agar.

5.2. OMP106 Polypeptide

OMP106 polypeptide of the invention is the sole outer membrane proteinof a HA M. catarrhalis strain or cultivar that has an apparent molecularweight in SDS-PAGE of about 180 kD to about 230 kD, preferably about 190kD. According to the invention, an outer membrane protein of M.catarrhalis is a polypeptide that is present in M. catarrhalis blebs, orthat can be extracted from M. catarrhalis blebs or intact cells byn-octyl β-D-glucopyranoside (OG) or sarkosyl detergent in buffersolution at room temperature. See Murphy and Loeb, 1989, MicrobialPathogenesis 6:159 174, for a discussion of M. catarrhalis blebs, whichare naturally occurring vesicles consisting of the outer membrane of M.catarrhalis. NHA M. catarrhalis strains or cultivars either do not haveOMP106 polypeptide, or have OMP106 polypeptide in a form that bindsanti-OMP106 antibodies (see Section 5.5., infra) but does not react withsilver stain (i.e., using Silver Stain Plus of BioRad [Richmond,Calif.], or the procedure described by Gottlieb and Chauko, 1987, Anal.Biochem. 165:33). By contrast, OMP106 polypeptide from HA M. catarrhalisstrains or cultivars binds anti-OMP106 antibodies, and reacts withsilver stain.

OMP106 polypeptide may be identified in HA M. catarrhalis blebs orintact cells by its susceptibility to degradation by protease treatmentthat also abolishes or attenuates the hemagglutination activity of thesame HA strain (See Section 5.1. above for examples of proteases that door do not destroy hemagglutination activity of intact M. catarrhaliscells). In other words, digestion with a protease that destroys orreduces the hemagglutination activity of a HA strain or cultivar willalso change, in SDS-PAGE, the abundance or the location of OMP106polypeptide isolated from the strain or cultivar after such a digestionas compared to that isolated from the same strain or cultivar before thedigestion.

OMP106 polypeptide may also be identified as the polypeptide in OG orsarkosyl extract of M. catarrhalis blebs or intact cells that has anapparent molecular weight of greater than 106 kD as determined bydenaturing gel electrophoresis in 12% PAG with SDS, using formulationsas described in Harlow and Lane (Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Appendix I,1988). Heat treatment of the OG or sarkosyl extract at 100° C. for 5minutes can cause the OMP106 polypeptide to have an apparent molecularweight of about 180 kD to about 230 kD as determined by SDS-PAGE in 6%PAG without any reducing agents, using formulations as described inHarlow and Lane, id. In a particular embodiment, OMP106 polypeptide inthe heat-treated OG or sarkosyl extract of M. catarrhalis strain ATCC®(American Type Culture Collection) 49143 has an apparent molecularweight of about 190 kD.

In particular embodiments, the OMP106 polypeptide is that prepared fromany of M. catarrhalis strains including, but not limited to, ATCC®(American Type Culture Collection) 49143, ATCC® (American Type CultureCollection) 25238, ATCC® (American Type Culture Collection) 25240, ATCC®(American Type Culture Collection) 43617, ATCC® (American Type CultureCollection) 43618, ATCC® (American Type Culture Collection) 43627 andATCC® (American Type Culture Collection) 43628. The preferred source ofOMP106 polypeptide is a HA cultivar of such strains. The more preferredsource is a HA cultivar of ATCC® (American Type Culture Collection)49143.

In a particular embodiment, OMP106 polypeptide comprises, preferably atthe amino-terminal, the amino acid sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or a sequencesubstantially homologous thereto. The OMP106 polypeptide mayadditionally comprise, carboxyl-distal to the above mentioned sequence,an octapeptide having the amino acid sequence GTVLGGKK (SEQ ID NO:2) ora sequence substantially homologous thereto. As used herein asubstantially homologous amino acid sequence is at least 80%, preferably100%, identical to the referenced amino acid sequence (see e.g. SEQ IDNO:11).

According to various aspects of the invention, the polypeptides of theinvention are characterized by their apparent molecular weights based onthe polypeptides' migration in SDS-PAGE relative to the migration ofknown molecular weight markers. While any molecular weight standardsknown in the art may be used with the SDS-PAGE, preferred molecularweight markers comprise at least rabbit skeletal muscle myosin, E. coliβ-galactosidase and rabbit muscle phosphorylase B. One skilled in theart will appreciate that the polypeptides of the invention may migratedifferently in different types of gel systems (e.g., different buffers;different concentration of gel, crosslinker or SDS). One skilled in theart will also appreciate that the polypeptides may have differentapparent molecular weights due to different molecular weight markersused with the SDS-PAGE. Hence, the molecular weight characterization ofthe polypeptides of the invention is intended to be directed to coverthe same polypeptides on any SDS-PAGE systems and with any molecularweight markers which might indicate slightly different apparentmolecular weights for the polypeptides than those disclosed here.

5.3. OMP106-Derived Polypeptides

An OMP106-derived polypeptide of the invention may be a fragment of theOMP106 polypeptide. The intact OMP106 polypeptide may contain one ormore amino acid residues that are not necessary to its immunogenicity.It may be the case, for example, that only the amino acid residuesforming a particular epitope of the OMP106 polypeptide is necessary forimmunogenic activity. Unnecessary amino acid sequences can be removed bytechniques well-known in the art. For example, the unwanted amino acidsequences can be removed by limited proteolytic digestion using enzymessuch as trypsin, papain, or related proteolytic enzymes or by chemicalcleavage using agents such as cyanogen bromide and followed byfractionation of the digestion or cleavage products.

An OMP106-derived polypeptide of the invention may also be a modifiedOMP106 polypeptide or fragment thereof (i.e., an OMP106 polypeptide orfragment having one or more amino acid substitutions, insertions and/ordeletions of the wild-type OMP106 sequence). Such modifications mayenhance the immunogenicity of the resultant polypeptide product or haveno effect on such activity. Modification techniques that may be usedinclude those disclosed in U.S. Pat. No. 4,526,716.

An OMP106-derived polypeptide may further be a chimeric polypeptidecomprising one or more heterologous polypeptides fused to theamino-terminal or carboxyl-terminal or internal of a complete OMP106polypeptide or a portion of or a fragment thereof. Useful heterologouspolypeptides comprising such chimeric polypeptide include, but are notlimited to, a) pre- and/or pro-sequences that facilitate the transport,translocation and/or processing of the OMP106-derived polypeptide in ahost cell, b) affinity purification sequences, and c) any usefulimmunogenic sequences (e.g., sequences encoding one or more epitopes ofa surface-exposed protein of a microbial pathogen).

Preferably, the OMP106-derived polypeptides of the invention areimmunologically cross-reactive with the OMP106 polypeptide, thus beingcapable of eliciting in an animal an immune response to M. catarrhalis.More preferably, the OMP106-derived polypeptides of the inventioncomprise sequences forming one or more outer-surface epitopes of thenative OMP106 polypeptide of M. catarrhalis (i.e., the surface-exposedepitopes of OMP106 polypeptide as it exists in intact M. catarrhaliscells). Such preferred OMP106-derived polypeptides can be identified bytheir ability to specifically bind antibodies raised to intact M.catarrhalis cells (e.g., antibodies elicited by formaldehyde orglutaldehyde fixed M. catarrhalis cells; such antibodies are referred toherein as “anti-whole cell” antibodies). For example, polypeptides orpeptides from a limited or complete protease digestion of the OMP106polypeptide are fractionated using standard methods and tested for theirability to bind anti-whole cell antibodies. Reactive polypeptidescomprise preferred OMP106-derived polypeptides. They are isolated andtheir amino acid sequences determined by methods known in the art.

Also preferably, the OMP106-derived polypeptides of the inventioncomprise sequences that form one or more epitopes of native OMP106polypeptide that mediate hemagglutination by HA M. catarrhalis cells.Such preferred OMP106-derived polypeptides may be identified by theirability to interfere with hemagglutination by HA M. catarrhalis cells.For example, polypeptides from a limited or complete protease digestionor chemical cleavage of OMP106 polypeptide are fractionated usingstandard methods and tested for the ability to interfere inhemagglutination by M. catarrhalis cells. Once identified and isolatedthe amino acid sequences of such preferred OMP106-derived polypeptidesare determined using standard sequencing methods. The determinedsequence may be used to enable production of such polypeptides bysynthetic chemical and/or genetic-engineering means.

These preferred OMP106-derived polypeptides also can be identified byusing anti-whole cell antibodies to screen bacterial librariesexpressing random fragments of M. catarrhalis genomic DNA or clonednucleotide sequences encoding the OMP106 polypeptide. See, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, N.Y., Vol. 1, Chapter 12. The reactive clones areidentified and their inserts are isolated and sequenced to determine theamino acid sequences of such preferred OMP106-derived polypeptides.

5.4. Isolation and Purification of OMP106 Polypeptide and OMP106-DerivedPolypeptides

The invention provides isolated OMP106 polypeptides and OMP106-derivedpolypeptides. As used herein, the term “isolated” means that the productis significantly free of other biological materials with which it isnaturally associated. That is, for example, an isolated OMP106polypeptide composition is between about 70% and 94% pure OMP106polypeptide by weight. Preferably, the OMP106 polypeptides andOMP106-derived polypeptides of the invention are purified. As usedherein, the term “purified” means that the product is substantially freeof other biological material with which it is naturally associated. Thatis comprising a purified OMP106 polypeptide composition is at least 95%pure OMP106 polypeptide by weight, preferably at least 98% pure OMP106polypeptide by weight, and most preferably at least 99% pure OMP106polypeptide by weight.

The OMP106 polypeptide of the invention may be isolated from proteinextracts including whole cell extract, of any M. catarrhalis strain orcultivar. Preferably, the protein extract is an octyl glucoside orsarkosyl extract of outer membrane vesicles (i.e., blebs) or whole cellsof M. catarrhalis including, but not limited to, any of strains ATCC®(American Type Culture Collection) 49143, ATCC® (American Type CultureCollection) 25238, ATCC® (American Type Culture Collection) 25240, ATCC®(American Type Culture Collection) 43617, ATCC® (American Type CultureCollection) 43618, ATCC® (American Type Culture Collection) 43627 andATCC® (American Type Culture Collection) 43628. The preferred source ofsuch extracts is a HA cultivar of such strains. The more preferredsource of such extracts is a HA cultivar of ATCC® (American Type CultureCollection) 49143. Another source of the OMP106 polypeptide is proteinpreparations from gene expression systems expressing cloned sequencesencoding OMP106 polypeptide or OMP106-derived polypeptides (see Section5.8., infra).

The OMP106 polypeptide can be isolated and purified from the sourcematerial using any biochemical technique and approach well known tothose skilled in the art. In one approach, M. catarrhalis outer membraneis obtained by standard techniques and outer membrane proteins aresolubilized using a solubilizing compound such as a detergent. Apreferred solubilizing solution is one containing about 1.25% octylglucopyranoside w/v (OG). Another preferred solubilizing solution is onecontaining about 1.25% sarkosyl. OMP106 polypeptide is in thesolubilized fraction. Cellular debris and insoluble material in theextract are separated and removed preferably by centrifuging. Thepolypeptides in the extract are concentrated, incubated inSDS-containing Laemmli gel sample buffer at 100° C. for 5 minutes andthen fractionated by electrophoresis in a 6% denaturing sodiumdodecylsulfate (SDS) polyacrylamide gel (PAG) without reducing agent.See Laemmli, 1970, Nature 227:680-685. The band or fraction identifiedas OMP106 polypeptide as described above (e.g., the silver-stainedpolypeptide band that is present in the OG or sarkosyl extract of a HAbut not that of a corresponding NHA cultivar or that of the HA cultivarafter digestion with a protease that abolishes hemagglutinationactivity) may then be isolated directly from the fraction or gel slicecontaining the OMP106 polypeptide. In a preferred embodiment, OMP106polypeptide has an apparent molecular weight of 190 kD as determined bycomparing its migration distance or rate in a denaturing SDS-PAGErelative to those of rabbit skeletal muscle myosin (200 kD) and E. coliβ-galactosidase (116 kD).

Another method of purifying OMP106 polypeptide is by affinitychromatography using anti-OMP106 antibodies, (see Section 5.5).Preferably, monoclonal anti-OMP106 antibodies are used. The antibodiesare covalently linked to agarose gels activated by cyanogen bromide orsuccinamide esters (Affi-Gel, BioRad, Inc.) or by other methods known tothose skilled in the art. The protein extract is loaded on the top ofthe gel as described above. The contact is for a period of time andunder standard reaction conditions sufficient for OMP106 polypeptide tobind to the antibody. Preferably, the solid support is a material usedin a chromatographic column. OMP106 polypeptide is then removed from theantibody, thereby permitting the recovery OMP106 polypeptide inisolated, or preferably, purified form.

An OMP106-derived polypeptide of the invention can be produced bychemical and/or enzymatic cleavage or degradation of isolated orpurified OMP106 polypeptide. An OMP106-derived polypeptide can also bechemically synthesized based on the known amino acid sequence of OMP106polypeptide and, in the case of a chimeric polypeptide, those of theheterologous polypeptide by methods well-known in the art. See, forexample, Creighton, 1983, Proteins: Structures and Molecular Principles,W.H. Freeman and Co., NY.

An OMP106-derived polypeptide can also be produced in a gene expressionsystem expressing a recombinant nucleotide construct comprisingsequences encoding OMP106-derived polypeptides. The nucleotide sequencesencoding polypeptides of the invention may be synthesized, and/orcloned, and expressed according to techniques well known to thoseskilled in the art. See, for example, Sambrook, et al., 1989, MolecularCloning, A Laboratory Manual, Vols. 1 3, Cold Spring Harbor Press, NY,Chapter 9.

OMP 106-derived polypeptides of the invention can be fractionated andpurified by the application of standard protein purification techniques,modified and applied in accordance with the discoveries and teachingsdescribed herein. In particular, preferred OMP106-polypeptides of theinvention, those that form an outer-surface epitope of the native OMP106polypeptide may be isolated and purified according to the affinityprocedures disclosed above for the isolation and purification of OMP106polypeptide (e.g., affinity purification using anti-OMP106 antibodies.

If desirable, the polypeptides of the invention may be further purifiedusing standard protein or peptide purification techniques including butare not limited to electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (including ion exchangechromatography, affinity chromatography, immunoadsorbent affinitychromatography, reverse-phase high performance liquid chromatography,and gel permeation high performance liquid chromatography), isoelectricfocusing, and variations and combinations thereof.

One or more of these techniques may be employed sequentially in aprocedure designed to separate molecules according to their physical orchemical characteristics. These characteristics include thehydrophobicity, charge, binding capability, and molecular weight of theprotein. The various fractions of materials obtained after eachtechnique are tested for their abilities to bind the OMP106 receptor orligand, to bind anti-OMP106 antibodies or to interfere withhemagglutination by HA M. catarrhalis cells (“test” activities). Thosefractions showing such activity are then subjected to the next techniquein the sequential procedure, and the new fractions are tested again. Theprocess is repeated until only one fraction having the above described“test” activities remains and that fraction produces only a single bandor entity when subjected to polyacrylamide gel electrophoresis orchromatography.

5.5. OMP106 Immunogens and Anti-OMP106 Antibodies

The present invention provides antibodies that specifically bind OMP106polypeptide or OMP106-derived polypeptides. For the production of suchantibodies, isolated or preferably, purified preparations of OMP106polypeptide or OMP106-derived polypeptides are used as immunogens.

In an embodiment, the OMP106 polypeptide is separated from other outermembrane proteins present in the OG or sarksyl extract of outer membraneof HA M. catarrhalis cells or blebs using SDS-PAGE (see Section 5.2.above) and the gel slice containing OMP106 polypeptide is used as theimmunogen and injected into a rabbit to produce antisera containingpolyclonal OMP106 antibodies. The same immunogen can be used to immunizemice for the production of hybridoma lines that produce monoclonalanti-OMP106 antibodies. In particular embodiments, a PAG slicecontaining isolated or purified OMP106 from any of strains ATCC®(American Type Culture Collection) 49143, ATCC® (American Type CultureCollection) 25238, ATCC® (American Type Culture Collection) 25240, ATCC®(American Type Culture Collection) 43617, ATCC® (American Type CultureCollection) 43618, ATCC® (American Type Culture Collection) 43627 andATCC® (American Type Culture Collection) 43628 is used as the immunogen.In preferred embodiments, a PAG slice containing isolated or purifiedOMP106 from a HA cultivar of such strains is used. In a more preferredembodiment, a PAG slice containing isolated or purified OMP106 from a HAcultivar of strain ATCC® (American Type Culture Collection) 49143 isused as the immunogen.

In other embodiments, peptide fragments of OMP106 polypeptide are usedas immunogens. Preferably, peptide fragments of purified OMP106polypeptide are used. The peptides may be produced by proteasedigestion, chemical cleavage of isolated or purified OMP106 polypeptideor chemical synthesis and then may be isolated or purified. Suchisolated or purified peptides can be used directly as immunogens. Inparticular embodiments, useful peptide fragments include but are notlimited to those having the sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or any portionthereof that is 6 or more amino acids in length. In an anotherembodiment, the peptide fragment has the sequence GTVLGGKK (SEQ IDNO:2).

Useful immunogens may also comprise such peptides or peptide fragmentsconjugated to a carrier molecule, preferably a carrier protein. Carrierproteins may be any commonly used in immunology, include, but are notlimited to, bovine serum albumin (BSA), chicken albumin, keyhole limpethemocyanin (KLH) and the like. For a discussion of hapten proteinconjugates, see, for example, Hartlow, et al., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988, or a standard immunology textbook such as Roitt, I. et al.,IMMUNOLOGY, C. V. Mosby Co., St. Louis, Mo. (1985) or Klein, J.,IMMUNOLOGY, Blackwell Scientific Publications, Inc., Cambridge, Mass.,(1990).

In yet another embodiment, for the production of antibodies thatspecifically bind one or more outer-surface epitopes of the nativeOMP106 polypeptide, intact HA M. catarrhalis cells or blebs preparedtherefrom are used as immunogen. The cells or blebs may be fixed withagents such as formaldehyde or glutaldehyde before immunization. SeeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988, Chapter 15. It ispreferred that such anti-whole cell antibodies be monoclonal antibodies.Hybridoma lines producing the desired monoclonal antibodies can beidentified by using purified OMP106 polypeptide as the screening ligand.Cells or blebs of any M. catarrhalis strain including, but not limitedto, ATCC® (American Type Culture Collection) 49143, ATCC® (American TypeCulture Collection) 25238, ATCC® (American Type Culture Collection)25240, ATCC® (American Type Culture Collection) 43617 ATCC® (AmericanType Culture Collection) 43618, ATCC® (American Type Culture Collection)43627 and ATCC® (American Type Culture Collection) 43628 are used as theimmunogen for inducing these antibodies. Preferably, cells or blebs of aHA cultivar of such strains are used as the immunogen. More preferably,cells or blebs of a HA cultivar of strain ATCC® (American Type CultureCollection) 49143 are used as the immunogen for inducing theseantibodies.

Polyclonal antibodies produced by whole cell or bleb immunizationscontain antibodies that bind other M. catarrhalis outer membraneproteins (“non-anti-OMP106 antibodies”) and thus are more cumbersome touse where it is known or suspected that the sample contains other M.catarrhalis outer membrane proteins or materials that are cross-reactivewith these other outer membrane proteins. Under such circumstances, anybinding by the anti-whole cell antibodies of a given sample or band mustbe verified by coincidental binding of the same sample or band byantibodies that specifically bind OMP106 polypeptide (e.g., anti-OMP106)and/or a OMP106-derived polypeptide, or by competition tests usinganti-OMP106 antibodies, OMP106 polypeptide or OMP106-derived polypeptideas the competitor (i.e., addition of anti-OMP106 antibodies, OMP106polypeptide or OMP106-derived polypeptide to the reaction mix lowers orabolishes sample binding by anti-whole cell antibodies). Alternatively,such polyclonal antisera, containing “non-anti-OMP106” antibodies, maybe cleared of such antibodies by standard approaches and methods. Forexample, the non-anti-OMP106 antibodies may be removed by precipitationwith cells of NHA M. catarrhalis cultivars or M. catarrhalis strainsknown not to have the OMP106 polypeptide (e.g., ATCC® (American TypeCulture Collection) 8176, more preferably a NHA cultivar of ATCC®(American Type Culture Collection) 49143); or by absorption to columnscomprising such cells or outer membrane proteins of such cells.

In further embodiments, useful immunogens for eliciting antibodies ofthe invention comprise mixtures of two or more of any of theabove-mentioned individual immunogens.

Immunization of mammals with the immunogens described herein, preferablyhumans, rabbits, rats, mice, sheep, goats, cows or horses, is performedfollowing procedures well known to those skilled in the art, forpurposes of obtaining antisera containing polyclonal antibodies orhybridoma lines secreting monoclonal antibodies.

Monoclonal antibodies can be prepared by standard techniques, given theteachings contained herein. Such techniques are disclosed, for example,in U.S. Pat. No. 4,271,145 and U.S. Pat. No. 4,196,265. Briefly, ananimal is immunized with the immunogen. Hybridomas are prepared byfusing spleen cells from the immunized animal with myeloma cells. Thefusion products are screened for those producing antibodies that bind tothe immunogen. The positive hybridomas clones are isolated, and themonoclonal antibodies are recovered from those clones.

Immunization regimens for production of both polyclonal and monoclonalantibodies are well-known in the art. The immunogen may be injected byany of a number of routes, including subcutaneous, intravenous,intraperitoneal, intradermal, intramuscular, mucosal, or a combinationof these. The immunogen may be injected in soluble form, aggregate form,attached to a physical carrier, or mixed with an adjuvant, using methodsand materials well-known in the art. The antisera and antibodies may bepurified using column chromatography methods well known to those ofskill in the art.

According to the present invention, OMP106 polypeptides of M.catarrhalis strains, HA or NHA, are immuno-cross reactive. Thus,antibodies raised to OMP106 polypeptide of one M. catarrhalis strain orcultivar specifically bind OMP106 polypeptide and OMP106-derivedpolypeptides of other M. catarrhalis strains and cultivars. For example,polyclonal anti-OMP106 antibodies induced by OMP106 polypeptide ofstrain ATCC® (American Type Culture Collection) 49143 specifically bindnot only the homologous OMP106 polypeptide (i.e., the OMP106 polypeptideof strain ATCC® (American Type Culture Collection) 49143) but alsoOMP106 polypeptide and/or OMP106-derived polypeptides of other M.catarrhalis strains including, but not limited to, ATCC® (American TypeCulture Collection) 43628, ATCC® (American Type Culture Collection)43627, ATCC® (American Type Culture Collection) 43618, ATCC® (AmericanType Culture Collection) 43617, ATCC® (American Type Culture Collection)25240 and ATCC® (American Type Culture Collection) 25238.

The antibodies of the invention, including but not limited toanti-OMP106 antibodies, can be used to facilitate isolation andpurification of OMP106 polypeptide and OMP106-derived polypeptides. Theantibodies may also be used as probes for identifying clones inexpression libraries that have inserts encoding OMP106 polypeptide orfragments thereof. The antibodies may also be used in immunoassays(e.g., ELISA, RIA, Westerns) to specifically detect and/or quantitate M.catarrhalis in biological specimens. Anti-OMP106 antibodies of theinvention specifically bind OMP106 polypeptide and do not bind proteinsfrom related bacterial pathogens such as Moraxella ovis, Moraxellalacunata, Moraxella osloensis, Moraxella bovis, Neisseria meningitidis,Neisseria gonorrhoeae. Thus anti-OMP106 antibodies can be used todiagnose M. catarrhalis infections.

The antibodies of the invention, particularly those which are cytotoxic,may also be used in passive immunization to prevent or attenuate M.catarrhalis infections of animals, including humans. (As used herein, acytotoxic antibody is one which enhances opsinization and/or complementkilling of the bacterium bound by the antibody) An effectiveconcentration of polyclonal or monoclonal antibodies raised against theimmunogens of the invention may be administered to a host to achievesuch effects. The exact concentration of the antibodies administeredwill vary according to each specific antibody preparation, but may bedetermined using standard techniques well known to those of ordinaryskill in the art. Administration of the antibodies may be accomplishedusing a variety of techniques, including, but not limited to thosedescribed in Section 5.6. for the delivery of vaccines.

Prophylactic and therapeutic efficacies of the antibodies of theinvention can be determined by standard pharmaceutical procedures inexperimental animals. The data obtained from animal studies can be usedin formulating a range of dosages for use in humans.

5.6. Vaccines

The present invention also provides therapeutic and prophylacticvaccines against M. catarrhalis infections of animals, includingmammals, and more specifically rodents, primates, and humans. Thepreferred use of the vaccines is in humans. The vaccines can be preparedby techniques known to those skilled in the art and would comprise, forexample, the antigen in form of an immunogen, a pharmaceuticallyacceptable carrier, possibly an appropriate adjuvant, and possibly othermaterials traditionally found in vaccines. An immunologically effectiveamount of the immunogen to be used in the vaccine is determined by meansknown in the art in view of the teachings herein.

The vaccines of the present invention comprise an immunologicallyeffective amount of any of the immunogens disclosed in Section 5.5. in apharmaceutically acceptable carrier.

According to another embodiment, the vaccines of the invention comprisean immunologically effective amount of an inactivated or attenuated HAM. catarrhalis cultivar or NHA M. catarrhalis cultivar of the invention.An inactivated or attenuated HA M. catarrhalis cultivar or NHA M.catarrhalis cultivar is obtained using any methods known in the artincluding, but not limited to, chemical treatment (e.g., formalin), heattreatment and irradiation.

The term “immunologically effective amount” is used herein to mean anamount sufficient to induce an immune response which can prevent M.catarrhalis infections or attenuate the severity of any preexisting orsubsequent M. catarrhalis infections. The exact concentration willdepend upon the specific immunogen to be administered, but may bedetermined by using standard techniques well known to those skilled inthe art for assaying the development of an immune response.

Useful polypeptide immunogens include the isolated OMP106 polypeptideand OMP106-derived polypeptides. Preferred immunogens include thepurified OMP106 polypeptide and derived polypeptides or peptides ofOMP106. The combined immunogen and carrier may be an aqueous solution,emulsion or suspension. In general, the quantity of polypeptideimmunogen will be between 0.1 and 500 micrograms per dose. The carriersare known to those skilled in the art and include stabilizers, diluents,and buffers. Suitable stabilizers include carbohydrates, such assorbitol, lactose, manitol, starch, sucrose, dextran, and glucose andproteins, such as albumin or casein. Suitable diluents include saline,Hanks Balanced Salts, and Ringers solution. Suitable buffers include analkali metal phosphate, an alkali metal carbonate, or an alkaline earthmetal carbonate. The vaccine may also contain one or more adjuvants toimprove or enhance the immunological response. Suitable adjuvantsinclude, but are not limited to, peptides; aluminum hydroxide; aluminumphosphate; aluminum oxide; a composition that consists of a mineral oil,such as Marcol 52, or a vegetable oil and one or more emulsifyingagents, or surface active substances such as lysolecithin, polycations,polyanions; and potentially useful human adjuvants such as BCG andCorynebacterium parvum. The vaccine may also contain other immunogens.Such a cocktail vaccine has the advantage that immunity against severalpathogens can be obtained by a single administration. Examples of otherimmunogens are those used in the known DPT vaccines.

The vaccines of the invention are prepared by techniques known to thoseskilled in the art, given the teachings contained herein. Generally, animmunogen is mixed with the carrier to form a solution, suspension, oremulsion. One or more of the additives discussed above may be in thecarrier or may be added subsequently. The vaccine preparations may bedesiccated, for example, by freeze drying for storage purposes. If so,they may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier.

The vaccines are administered to humans or other mammals, includingrodents and primates. They can be administered in one or more doses. Thevaccines may be administered by known routes of administration. Manymethods may be used to introduce the vaccine formulations describedhere. These methods include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. The preferred routes are intramuscular orsubcutaneous injection.

The invention also provides a method for inducing an immune response toM. catarrhalis in a mammal in order to protect the mammal againstinfection and/or attenuate disease caused by M. catarrhalis. The methodcomprises administering an immunologically effective amount of theimmunogens of the invention to the host and, preferably, administeringthe vaccines of the invention to the host.

5.7. Nucleic Acids Encoding OMP106 Polypeptide and OMP106-DerivedPolypeptides

The present invention also provides nucleic acids, DNA and RNA, encodingOMP106 polypeptide and OMP106-derived polypeptides. The nucleotidesequence of the entire OMP106 gene is depicted in SEQ ID NO:8. A deducedamino acid sequence of the open reading frame of OMP106 is depicted inSEQ ID NO:9.

In one aspect, the nucleic acids of the invention may be synthesizedusing methods known in the art. Specifically, a portion of or the entireamino acid sequence of OMP106 polypeptide or an OMP106-derivedpolypeptide may be determined using techniques well known to those ofskill in the art, such as via the Edman degradation technique (see,e.g., Creighton, 1983, Proteins: Structures and Molecular Principles,W.H. Freeman & Co., N.Y., pp. 34-49). The amino acid sequence obtainedis used as a guide for the synthesis of DNA encoding OMP106 polypeptideor OMP106-derived polypeptide using conventional chemical approaches orpolymerase chain reaction (PCR) amplification of overlappingoligonucleotides.

In another aspect, the amino acid sequence may be used as a guide forsynthesis of oligonucleotide mixtures which in turn can be used toscreen for OMP106 polypeptide coding sequences in M. catarrhalis genomiclibraries. Such libraries may be prepared by isolating DNA from cells ofany M. catarrhalis strain. Preferably the DNA used as the source of theOMP106 polypeptide coding sequence, for both genomic libraries and PCRamplification, is prepared from cells of any M. catarrhalis strainincluding, but not limited to ATCC® (American Type Culture Collection)49143, ATCC® (American Type Culture Collection) 25238, ATCC® (AmericanType Culture Collection) 25240, ATCC® (American Type Culture Collection)43617, ATCC® (American Type Culture Collection) 43618, ATCC® (AmericanType Culture Collection) 43627 and ATCC® (American Type CultureCollection) 43628.

In the preparation of genomic libraries, DNA fragments are generated,some of which will encode parts or the whole of M. catarrhalis OMP106polypeptide. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNase in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, column chromatographyand sucrose gradient centrifugation. The DNA fragments can then beinserted into suitable vectors, including but not limited to plasmids,cosmids, bacteriophages lambda or T₄, and yeast artificial chromosome(YAC). (See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) The genomic librarymay be screened by nucleic acid hybridization to labeled probe (Bentonand Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc.Natl. Acad. Sci. U.S.A. 72:3961).

The genomic libraries may be screened with a labeled degenerateoligonucleotide corresponding to the amino acid sequence of any peptideof OMP106 polypeptide using optimal approaches well known in the art. Inparticular embodiments, the screening probe is a degenerateoligonucleotide that corresponds to the peptide having the sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or a portionthereof. In another embodiment the screening probe may be a degenerateoligonucleotide that corresponds to a peptide having the sequenceGTVLGGKK (SEQ ID NO:2). In an additional embodiment, theoligonucleotides GGNACNGTNCTNGGNGGNAARAAR (SEQ ID NO:3) andGGNACNGTNTTRGGNGGNAARAAR (SEQ ID NO:7), each corresponding to OMP106peptide GTVLGGKK (SEQ ID NO:2), is used as the probe. In furtherembodiments, the sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCC® (American Type CultureCollection) ATTGCTATTGGTG ACATTGCGCAA (SEQ ID NO:4) or any fragmentsthereof, or any complement of the sequence or fragments may be used asthe probe. Any probe used preferably is 15 nucleotides or longer.

Clones in libraries with insert DNA encoding the OMP106 polypeptide orfragments thereof will hybridize to one or more of the degenerateoligonucleotide probes. Hybridization of such oligonucleotide probes togenomic libraries are carried out using methods known in the art. Forexample, hybridization with the two above-mentioned oligonucleotideprobes may be carried out in 2×SSC, 1.0% SDS at 50° C. and washed usingthe same conditions. In a particular embodiment, ATCC® (American TypeCulture Collection) 49143 DNA sequence encoding the whole or a part ofthe OMP106 polypeptide is a HindIII restriction fragment of about 8,000bp in length or a DRAI restriction fragment of about 4,200 bp in length.

In yet another aspect, clones of nucleotide sequences encoding a part orthe entire OMP106 polypeptide or OMP106-derived polypeptides may also beobtained by screening M. catarrhalis expression libraries. For example,M. catarrhalis DNA is isolated and random fragments are prepared andligated into an expression vector (e.g., a bacteriophage, plasmid,phagemid or cosmid) such that the inserted sequence in the vector iscapable of being expressed by the host cell into which the vector isthen introduced. Various screening assays can then be used to select forthe expressed OMP106 polypeptide or OMP106-derived polypeptides. In oneembodiment, the various anti-OMP106 antibodies of the invention (seeSection 5.5) can be used to identify the desired clones using methodsknown in the art. See, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., Appendix IV. Clones or plaques from the library arebrought into contact with the antibodies to identify those clones thatbind.

In an embodiment, colonies or plaques containing DNA that encodes OMP106polypeptide or OMP106-derived polypeptide could be detected using DYNABeads according to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. Anti-OMP106 antibodies are crosslinkedto tosylated DYNA Beads M280, and these antibody-containing beads wouldthen be used to adsorb to colonies or plaques expressing OMP106polypeptide or OMP106-derived polypeptide. Colonies or plaquesexpressing OMP106 polypeptide or OMP106-derived polypeptide isidentified as any of those that bind the beads.

Alternatively, the anti-OMP106 antibodies can be nonspecificallyimmobilized to a suitable support, such as silica or Celite™ resin. Thismaterial would then be used to adsorb to bacterial colonies expressingOMP106 polypeptide or OMP106-derived polypeptide as described in thepreceding paragraph.

In another aspect, PCR amplification may be used to producesubstantially pure DNA encoding a part of or the whole of OMP106polypeptide from M. catarrhalis genomic DNA. Oligonucleotide primers,degenerate or otherwise, corresponding to known OMP106 polypeptidesequences can be used as primers. In particular embodiments, anoligonucleotide, degenerate or otherwise, encoding the peptideIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or any portionthereof may be used as the 5′ primer. For example, a 5′ primer may bethe nucleotide sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCC® (American Type CultureCollection) ATTGCTATTGGTG ACATTGCGCAA (SEQ ID NO:4) or any portionthereof. Nucleotide sequences, degenerate or otherwise, that are reversecomplements of sequence encoding GTVLGGKK (SEQ ID NO:2) may be used asthe 3′ primer.

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). One can choose to synthesizeseveral different degenerate primers, for use in the PCR reactions. Itis also possible to vary the stringency of hybridization conditions usedin priming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence similarity between the degenerate primers and thecorresponding sequences in M. catarrhalis DNA. After successfulamplification of a segment of the sequence encoding OMP106 polypeptide,that segment may be molecularly cloned and sequenced, and utilized as aprobe to isolate a complete genomic clone. This, in turn, will permitthe determination of the gene's complete nucleotide sequence, theanalysis of its expression, and the production of its protein productfor functional analysis, as described infra.

Once an OMP106 polypeptide coding sequence has been isolated from one M.catarrhalis strain or cultivar, it is possible to use the same approachto isolate OMP106 polypeptide coding sequences from other M. catarrhalisstrains and cultivars. It will be recognized by those skilled in the artthat the DNA or RNA sequence encoding OMP106 polypeptide (or fragmentsthereof) of the invention can be used to obtain other DNA or RNAsequences that hybridize with it under conditions of moderate to highstringency, using general techniques known in the art. Hybridizationwith an OMP106 sequence from one M. catarrhalis strain or cultivar underhigh stringency conditions will identify the corresponding sequence fromother strains and cultivars. High stringency conditions vary with probelength and base composition. The formula for determining such conditionsare well known in the art. See Sambrook et al., 1989, Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Press, NY, Chapter 11. As usedherein high stringency hybridization conditions as applied to probes ofgreater than 300 bases in length involve a final wash in 0.1×SSC/0.1%SDS at 68° C. for at least 1 hour (Ausubel, et al., Eds., 1989, CurrentProtocols in Molecular Biology, Vol. I, Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., New York, at page 2.10.3). Inparticular embodiments, the high stringency wash in hybridization usinga probe having the sequence of SEQ ID NO:4 or its complement is 2×SSC,1% SDS at 50° C. for about 20 to about 30 minutes.

One skilled in the art would be able to identify complete clones ofOMP106 polypeptide coding sequence using approaches well known in theart. The extent of OMP106 polypeptide coding sequence contained in anisolated clone may be ascertained by sequencing the cloned insert andcomparing the deduced size of the polypeptide encoded by the openreading frames (ORFs) with that of OMP106 polypeptide and/or bycomparing the deduced amino acid sequence with that of known amino acidsequence of purified OMP106 polypeptide. Where a partial clone of OMP106polypeptide coding sequence has been isolated, complete clones may beisolated by using the insert of the partial clone as hybridizationprobe. Alternatively, a complete OMP106 polypeptide coding sequence canbe reconstructed from overlapping partial clones by splicing theirinserts together.

Complete clones may be any that have ORFs with deduced amino acidsequence matching that of OMP106 polypeptide or, where the completeamino acid sequence of the latter is not available, that of a peptidefragment of OMP106 polypeptide and having a molecular weightcorresponding to that of OMP106 polypeptide. Further, complete clonesmay be identified by the ability of their inserts, when placed in anexpression vector, to produce a polypeptide that binds antibodiesspecific to the amino-terminal of OMP106 polypeptide and antibodiesspecific to the carboxyl-terminal of OMP106 polypeptide.

Nucleic acid sequences encoding OMP106-derived polypeptides may beproduced by methods well known in the art. In one aspect, sequencesencoding OMP106-derived polypeptides can be derived from OMP106polypeptide coding sequences by recombinant DNA methods in view of theteachings disclosed herein. For example, the coding sequence of OMP106polypeptide may be altered creating amino acid substitutions that willnot affect the immunogenicity of the OMP106 polypeptide or which mayimprove its immunogenicity. Various methods may be used, including butnot limited to oligonucleotide directed, site specific mutagenesis.These and other techniques known in the art may be used to create singleor multiple mutations, such as replacements, insertions, deletions, andtranspositions, as described in Botstein and Shortle, 1985, Science229:1193 1210.

Further, DNA of OMP106 polypeptide coding sequences may be truncated byrestriction enzyme or exonuclease digestions. Heterologous codingsequence may be added to OMP106 polypeptide coding sequence by ligationor PCR amplification. Moreover, DNA encoding the whole or a part of anOMP-derived polypeptide may be synthesized chemically or using PCRamplification based on the known or deduced amino acid sequence ofOMP106 polypeptide and any desired alterations to that sequence.

The identified and isolated DNA containing OMP106 polypeptide orOMP106-derived polypeptide coding sequence can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini. However,if the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved DNA may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired DNA containing OMP106 polypeptideor OMP106-derived polypeptide coding sequence may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired sequence, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that contain OMP106 polypeptide or OMP106-derivedpolypeptide coding sequence enables generation of multiple copies ofsuch coding sequence. Thus, the coding sequence may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted coding sequence from the isolated recombinant DNA.

5.8. Recombinant Production of OMP106 Polypeptide and OMP106-DerivedPolypeptides

OMP106 polypeptide and OMP106-derived polypeptides of the invention maybe produced through genetic engineering techniques. In this case, theyare produced by an appropriate host cell that has been transformed byDNA that codes for the polypeptide. The nucleotide sequence encodingOMP106 polypeptide or OMP106-derived polypeptides of the invention canbe inserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted polypeptide-coding sequence. The nucleotide sequencesencoding OMP106 polypeptide or OMP106-derived polypeptides is insertedinto the vectors in a manner that they will be expressed underappropriate conditions (e.g., in proper orientation and correct readingframe and with appropriate expression sequences, including an RNApolymerase binding sequence and a ribosomal binding sequence).

A variety of host-vector systems may be utilized to express thepolypeptide-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.Preferably, the host cell is a bacterium, and most preferably thebacterium is E. coli, B. subtilis or Salmonella.

The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In a specific embodiment, a chimeric protein comprising OMP106polypeptide or OMP106-derived polypeptide sequence and a pre and/or prosequence of the host cell is expressed. In other specific embodiments, achimeric protein comprising OMP106 polypeptide or OMP106-derivedpolypeptide sequence and an affinity purification peptide is expressed.In further specific embodiments, a chimeric protein comprising OMP106polypeptide or OMP106-derived polypeptide sequence and a usefulimmunogenic peptide or polypeptide is expressed. In preferredembodiments, OMP106-derived polypeptide expressed contains a sequenceforming either an outer-surface epitope or the receptor-binding domainof native OMP106 polypeptide.

Any method known in the art for inserting DNA fragments into a vectormay be used to construct expression vectors containing a chimeric geneconsisting of appropriate transcriptional/translational control signalsand the polypeptide coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombinants(genetic recombination). Expression of a nucleic acid sequence encodingOMP106 polypeptide or OMP106-derived polypeptide may be regulated by asecond nucleic acid sequence so that the inserted sequence is expressedin a host transformed with the recombinant DNA molecule. For example,expression of the inserted sequence may be controlled by anypromoter/enhancer element known in the art. Promoters which may be usedto control expression of inserted sequences include, but are not limitedto the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42) for expression in animalcells; the promoters of β-lactamase (Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:21-25), λP_(L), or trc for expression inbacterial cells (see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94); the nopaline synthetase promoterregion or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,1981, Nucl. Acids Res. 9:2871), and the promoter of the photosyntheticenzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984,Nature 310:115-120) for expression implant cells; promoter elements fromyeast or other fungi such as the Gal4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter.

Expression vectors containing OMP106 polypeptide or OMP106-derivedpolypeptide coding sequences can be identified by three generalapproaches: (a) nucleic acid hybridization, (b) presence or absence of“marker” gene functions, and (c) expression of inserted sequences. Inthe first approach, the presence of a foreign gene inserted in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to the inserted OMP106polypeptide or OMP106-derived polypeptide coding sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign genes in the vector. For example, ifthe OMP106 polypeptide or OMP106-derived polypeptide coding sequence isinserted within the marker gene sequence of the vector, recombinantscontaining the insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the foreign gene product expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of OMP106 polypeptide or OMP106-derivedpolypeptide in in vitro assay systems, e.g., binding to an OMP106 ligandor receptor, or binding with anti-OMP106 antibodies of the invention, orthe ability of the host cell to hemagglutinate or the ability of thecell extract to interfere with hemagglutination by M. catarrhalis.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Asexplained above, the expression vectors which can be used include, butare not limited to, the following vectors or their derivatives: human oranimal viruses such as vaccinia virus or adenovirus; insect viruses suchas baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), andplasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered OMP106 polypeptide orOMP106-derived polypeptide may be controlled. Furthermore, differenthost cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe desired modification and processing of the foreign proteinexpressed.

5.9. Reagents

The polypeptides, peptides, antibodies and nucleic acids of theinvention are useful as reagents for clinical or medical diagnosis of M.catarrhalis infections and for scientific research on the properties ofpathogenicity, virulence, and infectivity of M. catarrhalis, as well ashost defense mechanisms. For example, DNA and RNA of the invention canbe used as probes to identify the presence of M. catarrhalis inbiological specimens by hybridization or PCR amplification. The DNA andRNA can also be used to identify other bacteria that might encode apolypeptide related to the M. catarrhalis OMP106.

The polypeptides and peptides of the invention may be used to preparepolyclonal and monoclonal antibodies that can be used to further purifycompositions containing the polypeptides of the invention by affinitychromatography. The polypeptides and peptides can also be used instandard immunoassays to screen for the presence of antibodies to M.catarrhalis in a sample.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and processes for their preparation and use appear in thefollowing example.

6. Example Isolation and Characterization of the OMP106 Polypeptide AndGene Encoding Same from Strain ATCC® (American Type Culture Collection)49143 or Other Strains 6.1. Material and Methods

6.1.1. Hemagglutination Assay

Hemagglutination by M. catarrhalis was tested as described bySoto-Hernandez et al. (J. Clin. Microbiol. 27:903-908) except 5%,instead of 3%, v/v erythrocytes were used in a slide agglutinationassay. Initial hemagglutination assays were performed using 20 μg ofbacterial cells (wet weight). Since M. catarrhalis ATCC® (American TypeCulture Collection) strain 49143 grown on blood agar plates at 35° C.gave a strong hemagglutination reaction, it was selected as thereference strain. Serially diluting ATCC® (American Type CultureCollection) strain 49143 in 1:2 dilutions resulted in decreasinghemagglutination reactions. Scores of ++++ to + were based on thehemagglutination observed by ATCC® (American Type Culture Collection)strain 49143 after serial 1:2 dilutions so that a + reaction resultedusing ¼ the number of cells required to achieve a +++ reaction.

6.1.2. Inhibition of Hemagglutination

M. catarrhalis ATCC® (American Type Culture Collection) 49143 cellsuspension was serially diluted 1:2, and the dilution that yielded a+hemagglutination reaction when 7 μl of Dulbecco's phosphate bufferedsaline and 7 μl of 5% (v/v) human O⁺ erythrocytes was used to assayinhibition of hemagglutination by simple sugars and sugar derivatives.To determine if simple sugars or sugar derivatives could inhibithemagglutination by M. catarrhalis, 7 μl of a given sugar at 500 mM wasmixed with 7 μl of M. catarrhalis cells and incubated for 5 minutes toallow the sugar to interact with the cells. Then 7 μl of 5% (v/v) humanO⁺ erythrocytes were added and the hemagglutination was scored after 1minute. Each sugar and sugar derivative was tested for the ability toinhibit hemagglutination. Then the stock of each sugar and sugarderivative was serially diluted 1:2, and these dilutions were assayedfor their ability to inhibit hemagglutination using the proceduredescribed above. In this manner, the minimal concentration ofcarbohydrate required to inhibit hemagglutination was determined.

6.1.3. Ligand and Receptor Binding

M. catarrhalis binding to animal cell glycolipid receptors was examinedusing thin layer chromatography (TLC) fractionation of the host cellglycolipids and labeled cell overlay of the chromatogram following theprocedures described by Magnani et al., 1982, J. Biol. Chem.257:14365-14369. Briefly, glycolipids obtained from Matreya Inc.(Pleasant Gap, Pa.) were resolved on high performance thin layerchromatograph plates (HPTLC) in chloroform, methanol, water (5:4:1) Theplates were either stained with orcinol at 100° C., or were overlaidwith ¹²⁵I-labeled M. catarrhalis blebs prepared as previously described(Murphy and Loeb, 1989, Microbial Pathogen. 6:159-174) at 2×10⁶ cpm/mlfor 2 hours. The plates were then washed 5 times, dried and exposed toX-ray film.

6.1.4. OG Extraction of OMPS

Strains of M. catarrhalis were each grown at 35° C. at 200 rpm in 1liter of Mueller Hinton broth in a 4 liter flask. Outer membrane protein(OMP) preparations were isolated by treating 50 mg of cells (wet weight)with 0.67 ml of 1.25% n-octyl β-D-glucopyranoside (i.e., octylglucoside; OG) in phosphate buffered saline (PBS) for 30 minutes at roomtemperature. Cells were pelleted in a microcentrifuge for 5 minutes andthe supernatant was used as an octyl glucoside extract. Comparison ofprotein profiles of these extracts from a number of strains of M.catarrhalis to those of blebs (i.e., outer membrane vesicles) isolatedby differential centrifugation, which are highly enriched for outermembrane proteins (OMPs) from M. catarrhalis (Murphy and Loeb, 1989,Microbial Pathogen. 6:159-174) indicates the octyl glucoside extractscontain predominately outer membrane proteins of M. catarrhalis (FIG.1). This indicated that octyl glycoside extraction provided a more rapidprocedure with a higher yield of outer membrane proteins as compared toouter membrane proteins prepared from blebs.

6.1.5. Proteolytic Digestion of OMP106

50 mg of cells from ATCC® (American Type Culture Collection) strain49143 in 1 ml of Dulbecco's phosphate buffered saline were digested for1 hour at room temperature with the following proteases: TLCK-treatedchymotrypsin (5 mg), Proteinase K (5 mg), TPCK-treated trypsin (5 mg),or protease V8 (100 Units). All proteases were obtained from SigmaChemicals (St. Louis, Mo.). Immediately following the proteasetreatment, cells were washed once in PBS and resuspended in 1 ml of PBSand the hemagglutinating activity was tested. Additionally,protease-treated bacterial cells were extracted with octyl glucoside sothe outer membrane proteins could be resolved to identify specificproteins that may have been digested by the proteases.

6.1.6. Non-Hemagglutinating Cultivars

Normally, hemagglutinating M. catarrhalis cultures are grown in shakerflasks containing Mueller Hinton Broth at 35 to 37° C. at 200 rpm for 24to 48 hours. Cells taken directly from a blood agar plate or an agarplate of Mueller Hinton media also express the hemagglutinatingphenotype. To select for a non-hemagglutinating (NHA) cultivar, ATCC®(American Type Culture Collection) strain 49143 was serially passagedevery 5 days in static cultures grown in Mueller Hinton broth at 35° C.With each passage, inoculum was taken only from the surface of the brothculture.

By the second passage, a floating mat of cells had developed and thismat of cells was used as the inoculum for subsequent cultures. Serialculturing in this manner produced NHA cultivars of ATCC® (American TypeCulture Collection) 49143 typically after three passages.

5 6.1.7. Isolation of OMP106 Polypeptide

OMP106 polypeptide from outer membrane extract of M. catarrhalis ATCC®(American Type Culture Collection) 49143 is detected (e.g., by silverstaining or anti-OMP106 antibodies) in denaturing gels only after theextract has been incubated at 100° C. for five minutes. In order todetermine if the appearance of the OMP106 band after incubation at 100°C. is the result of lower molecular weight proteins aggregating duringboiling, or if the boiling allows a normally aggregated protein to enterthe gel, an unboiled octyl glucoside outer membrane extract of ATCC®(American Type Culture Collection) 49143 was analyzed on a nativepolyacrylamide gel. Specific regions of the gel including thatimmediately below the sample well were excised and boiled. The resultingsamples were then resolved on a denaturing polyacrylamide gel andstained with silver stain (Silver Stain Plus, Catalog number 161-0449,BioRad Laboratories, Richmond, Calif.). For N-terminal sequencing, anoctyl glucoside outer membrane extract of ATCC® (American Type CultureCollection) 49143 was mixed with PAGE sample buffer containing SDS, andwas incubated for 5 minutes in boiling water bath. The proteins werethen resolved on a 12% PAG with SDS and transferred to a PVDF membraneby electroblotting. The region of the membrane containing the OMP106band was then cut out for amino-terminal sequencing. None of the PAGEprocedures used to isolate the OMP106 polypeptide used reducing agentsin the sample or gel buffers.

6.1.8. Anti-OMP106 Antiserum

Antiserum to OMP106 were prepared by resolving OMP106 polypeptide from aHA cultivar of ATCC® (American Type Culture Collection) 49143 in adenaturing sodium dodecylsulfate polyacrylamide gel as previouslydescribed (Lammeli, 1970, Nature 227:680-685), and cutting theOMP106-containing band out of the gel. The excised band was maceratedand injected into a rabbit to generate antiserum to OMP106 polypeptide.The antiserum was used to inhibit hemagglutination as described insection 6.1.2. supra, but using the antiserum in place of thecarbohydrate. The antiserum was also examined for complement-mediatedcytotoxic activity against M. catarrhalis as described in section 7.

6.1.9. Western Blots with Anti-OMP106 Antiserum

M. catarrhalis ATCC® (American Type Culture Collection) 49143, ATCC®(American Type Culture Collection) 43628, ATCC® (American Type CultureCollection) 43627, ATCC® (American Type Culture Collection) 43618 ATCC®(American Type Culture Collection) 43617, ATCC® (American Type CultureCollection) 25240, ATCC® (American Type Culture Collection) 25238, andATCC® (American Type Culture Collection) 8176; M. ovis ATCC® (AmericanType Culture Collection) 33078; M. lacunta ATCC® (American Type CultureCollection) 17967; M. bovis ATCC® (American Type Culture Collection)10900; M. osloensis ATCC® (American Type Culture Collection) 10973;Neisseria gonorrhoeae (clinical isolate); and N. meningitidis ATCC®(American Type Culture Collection) 13077 were grown on chocolate agarplates for 48 hours at 35° C. in 5% CO₂. Cells were removed by scrapingthe colonies from the agar surface using a polystyrene inoculating loop.Cells were then solubilized by suspending 30 μg of cells in 150 μl ofPAGE sample buffer (360 mM Tris buffer [pH 8.8], containing 4% sodiumdodecylsulfate and 20% glycerol), and incubating the suspension at 100°C. for 5 minutes. The solubilized cells were resolved on 12%polyacrylamide gels as per Laemmli and the separated proteins wereelectrophoretically transferred to PVDF membranes at 100 V for 1.5 hoursas previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci.USA 76:4350-4354) except 0.05% sodium dodecylsulfate was added to thetransfer buffer to facilitate the movement of proteins from the gel. ThePVDF membranes were then pretreated with 25 ml of Dulbecco's phosphatebuffered saline containing 0.5% sodium casein, 0.5% bovine serum albuminand 1% goat serum. All subsequent incubations were carried out usingthis pretreatment buffer.

PVDF membranes were incubated with 25 ml of a 1:500 dilution ofpreimmune rabbit serum or serum from a rabbit immunized with OMP106polypeptide (as described above) for 1 hour at room temperature. PVDFmembranes were then washed twice with wash buffer (20 mM Tris buffer [pH7.5.] containing 150 mM sodium chloride and 0.05% Tween-20). PVDFmembranes were incubated with 25 ml of a 1:5000 dilution ofperoxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearchLaboratories, West Grove Pa. Catalog number 111-035-003) for 30 minutesat room temperature. PVDF membranes were then washed 4 times with washbuffer, and were developed with 3,3′ diaminobenzidine tetrahydrochlorideand urea peroxide as supplied by Sigma Chemical Co. (St. Louis, Mo.catalog number D-4418) for 4 minutes each.

6.1.10. Anti-OMP106 Immunofluorescence Staining of Cell Surface

M. catarrhalis ATCC® (American Type Culture Collection) 49143 was grownovernight at 35° C. in a shaking water bath in Mueller Hinton broth. Thecells were pelleted by centrifugation and then resuspended in an equalvolume of Dulbecco's modification of phosphate buffered saline withoutcalcium or magnesium (PBS/MC). 20 μl of the cell suspension was appliedto each of 5 clean microscope slides. After setting for 10 seconds, theexcess fluid was removed with a micropipettor, and the slides wereallowed to air dry for 1 hour. The slides were then heat fixed over anopen flame until the glass was warm to the touch. The slides wereinitially treated with 40 μl of 1:40 dilution of anti-OMP106 antiserumor preimmune serum from the same animal diluted in PBS/MC, or PBS/MC for10 minutes, then washed 5 times with PBS/MC. The slides were treatedwith 40 μl of 5 μg/ml PBS/MC of fluorescein isothiocyanate-labeled goatantibody to rabbit IgG (Kirkegaard and Perry Laboratories, Inc,Gaithersburg, Md. catalog number 02-15-06). The slides were incubated inthe dark for 10 minutes and were washed 5 times in PBS/MC. Each slidewas stored covered with PBS/MC under a cover slide and was viewed with afluorescence microscope fitted with a 489 nm filter. For each samplefive fields-of-view were visually examined to evaluate the extent ofstraining.

6.2. Results

6.2.1. Hemagglutination Activity

The agglutination activity of M. catarrhalis with respect toerythrocytes is species specific with the strongest activity observedwith human erythrocytes. Rabbit erythrocytes are also agglutinated by M.catarrhalis, but less dramatically than are human cells. Theerythrocytes from mouse, horse or sheep were not agglutinated (see Table1).

TABLE 1 Strength of hemagglutination of erythrocytes from variousspecies using M. catarrhalis ATCC ® (American Type Culture Collection)49143 Source of Score for erythrocytes Source of Score forhemagglutination^(a) Human ++++ Rabbit ++ Mouse − Horse − Sheep −

6.2.2. OMP106 Receptors and Ligands

M. catarrhalis hemagglutination activity is due to binding toglobotetrose (Gb₄). Blebs from hemagglutinating strains bind to aglycolipid having Gb₄, whereas non-hemagglutinating strains do not bindto the same glycolipid (see FIG. 2). M. catarrhalis hemagglutinationactivity is inhibited by monosaccharide constituents of Gb₄ orderivatives of such monosaccharides, with the most potent inhibitorsbeing N-acetyl galactosamine and galactose (especially the alpha anomerof the galactose) (see Table 2).

TABLE 2 The minimum concentration of sugars required to inhibithemagglutination (MIC) by M. catarrhalis Sugar MIC (mM)* D-Glucose >167D-Mannose 83 D-Galactose 41 L-Fucose 83 N-acetyl-D-Glucosamine >167N-acetyl-D-Galactosamine 41 Methyl-α-Glucose >167 Methyl-α-Mannose 167Methyl-α-Galactose 10 Methyl-β-galactose 83 *Minimal concentration ofsugar required to inhibit a 1+ hemagglutination reaction by M.catarrhalis ATCC ® (American Type Culture Collection) 49143 with 5%washed human O+ erythrocytes.

Both N-acetyl galactosamine and alpha-galactose are part of the Gb₄tetrasaccharide. The correlation between hemagglutination and binding toGb₄, and the observation that hemagglutination is inhibited bymonosaccharides that comprise the Gb₄ receptor suggest that M.catarrhalis cells bind to the tetrasaccharide Gb₄. This tetrasaccharideis present on human erythrocytes and tissues, and could mediate M.catarrhalis attachment to eukaryotic membranes.

6.2.3. Identification of OMP106 Polypeptide

Proteolytic digestion of M. catarrhalis cells, and subsequent analysisof hemagglutination by the digested cells demonstrated that proteasetreatment with chymotrypsin and proteinase K destroyed thehemagglutination activity, and treatment with trypsin partiallydestroyed hemagglutination activity, indicating the hemagglutinatingactivity is protein mediated. Analysis of the OMP protein profiles ofprotease digested M. catarrhalis cells showed that multiple proteins hadbeen degraded in each sample, so the profiles did not provide a clue asto which protein is directly responsible for or indirectly contributedto the hemagglutination activity (see FIG. 3).

Since protease treatment indicated a polypeptide is directly orindirectly responsible for hemagglutination activity, we used SDS-PAGEto compare the OMP profiles from hemagglutinating strains with the OMPprofiles from non-hemagglutinating strains (FIG. 4). Analysis of thedifferences between these profiles indicated that the hemagglutinatingstrains had two unique polypeptides, one with an apparent molecularweight of 27 kD (designated OMP27) and the other was the only proteinwith an apparent molecular weight of greater than 106 kD (designatedOMP106). Notably, the OMP106 polypeptide band was absent in the OMPpreparations of various protease treated cells that have reduced or nohemagglutination activity, whereas the OMP27 band was present in the OMPpreparation of proteinase K treated cells that have no hemagglutinationactivity. Additionally, the OMP106 polypeptide band was not degraded byproteinase V8 digestion, which did not affect hemagglutination activityof treated cells.

6.2.4. OMP Profile of NHA Cultivars

Serial culturing of NHA cultivar of ATCC® (American Type CultureCollection) 49143 in static culture at 35° C. produced a NHA cultivar(designated 49143-NHA) by the third passage of the culture. This loss ofthe hemagglutination activity was repeatable. Analysis of OMP profilesof OG outer membrane extracts of the HA and NHA cultivars showed thatthe OMP106 polypeptide band was missing from the 49143-NHA extract (FIG.5). This suggested that OMP106 polypeptide is the M. catarrhalishemagglutinin (i.e., OMP106 polypeptide binds Gb₄ receptor or is asubunit of a homopolymeric protein that binds Gb₄ receptor) or forms apart of the M. catarrhalis hemagglutinin (i.e., OMP106 polypeptide is asubunit of a heteropolymeric protein that binds Gb₄ receptor).

6.2.5. OMP106 and Hemagglutination

Polyclonal antiserum raised to ATCC® (American Type Culture Collection)49143 OMP106 polypeptide neutralized hemagglutination by ATCC® (AmericanType Culture Collection) 49143, as well as that by heterologous ATCC®(American Type Culture Collection) 43627. This further supports theconclusion that M. catarrhalis hemagglutinating activity comprisesOMP106 polypeptide, and that OMP106 polypeptide is antigenicallyconserved among strains. See also FIG. 9A, which shows antibodies in thepolyclonal antiserum binding OMP106 polypeptide of heterologous M.catarrhalis strains.

6.2.6. Outer Surface Location of OMP106

Rabbit anti-OMP106 antiserum was used in indirect immunofluorescencestaining to determine if OMP106 polypeptide is exposed on the outersurface of M. catarrhalis cells. M. catarrhalis cells treated withanti-OMP106 antiserum stained more intensely and uniformly than didcells treated with preimmune serum or PBS/MC. This indicated that inintact M. catarrhalis cells OMP 106 polypeptide was reactive withanti-OMP106 antibodies. This result indicates that OMP106 polypeptide isexposed on the outer surface of M. catarrhalis. This finding isconsistent with OMP106 polypeptide having a role in hemagglutinationand, moreover, indicates that OMP106 polypeptide would be useful as avaccine.

6.2.7. Properties of OMP106 Polypeptide

OMP106 polypeptide exists as a large protein complex in its native stateor aggregates when extracted with octyl glucoside. This conclusion issupported by the finding that extracting M. catarrhalis cells with octylglucoside will solubilize OMP106 polypeptide, but the extracted OMP106polypeptide does not enter denaturing PAGs unless the extract is firstincubated at 100° C. (FIG. 6). Further, the OMP106 polypeptide band doesnot appear to form from lower molecular weight polypeptides thatpolymerize or aggregate upon heating, since OMP106 polypeptide in anon-heat denatured sample is trapped in the sample well and enters theresolving gel only if the sample has been first incubated at 100° C.This biochemical property is very useful for identifying OMP106polypeptide in various gels.

Using octyl glucoside extracts of M. catarrhalis, then incubating theextracts with sodium dodecyl sulfate at 100° C., and resolving theproteins on a denaturing polyacrylamide gel, we have estimated theapparent molecular weight of OMP106 polypeptide from various strains ofM. catarrhalis, specifically those of ATCC® (American Type CultureCollection) 25238, ATCC® (American Type Culture Collection) 25240, ATCC®(American Type Culture Collection) 43617, ATCC® (American Type CultureCollection) 43618, ATCC® (American Type Culture Collection) 43627 andATCC® (American Type Culture Collection) 43628, to range from about 180kD to about 230 kD (FIG. 9A), whereas the OMP106 polypeptide of strainATCC® (American Type Culture Collection) 49143 appears to have anapparent weight of about 190 kD (FIG. 6).

OMP106 polypeptide of strain ATCC® (American Type Culture Collection)49143 was extracted from the gel slice and was sequenced. N-terminalsequencing of the mature OMP106 polypeptide isolated from the outermembrane of ATCC® (American Type Culture Collection) 49143 yielded thefollowing sequence: IGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ IDNO:1). Further analysis of the nucleotide sequence encoding the matureOMP106 protein demonstrated that the amino terminal sequence is:GIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGD (SEQ ID NO:11) Additionally, aninternal peptide of OMP106 produced by digestion with Lys-C (Fernandezet al., 1994, Anal Biochem 218:112-117) has been isolated and yieldedthe following sequence: GTVLGGKK (SEQ ID NO:2).

We generated three oligonucleotide probes. Two probes correspond to theinternal peptide GTVLGGKK, one has the following sequenceGGNACNGTNCTNGGNGGNAARAAR (SEQ ID NO:3), the other has the followingsequence GGNACNGTNTTRGGNGGNAARAAR (SEQ ID NO:7). The other probe, Mc5-72, encoding an internal fragment (SEQ ID NO:5) of the amino-terminalsequence of OMP106 (SEQ ID NO:1) has the following sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCC® (American Type CultureCollection) ATTGCTATTGGTG ACATTGCGCAA (SEQ ID NO:4). Hybridization ofthe Mc 5-72 probe to a complete HindIII or DraI digest of M. catarrhalisDNA in each instance produced a single band in Southern blot analysis(FIG. 7). The hybridizing band in the HindIII digest has an approximatesize of 8.0 kb; the hybridizing band in the DraI digest has anapproximate size of 4.2 kb (FIG. 7).

6.2.8. Conservation of OMP106 Polypeptide

Western blot analysis of outer membrane protein extracts of a number ofM. catarrhalis strains and related species of bacteria showed that theanti-OMP106 antibodies binds to a polypeptide of about 180 Kd to about230 kD in many M. catarrhalis strains, both HA and NHA strains orcultivars (FIG. 9A). The anti-OMP106 antibodies did not bind to anypolypeptide in the protein extracts of related bacteria (FIG. 8A). Theseresults demonstrate the following: 1) Anti-OMP106 antibodies may be usedto specifically identify and distinguish M. catarrhalis from relatedspecies of bacteria. 2) OMP106 polypeptide may be used to generateantibodies that have diagnostic application for identification of M.catarrhalis. 3) Antibodies to OMP106 polypeptide of one strain (e.g.,OMP106 of ATCC® (American Type Culture Collection) 49143) may be used toidentify and isolate the corresponding OMP106 polypeptide of other M.catarrhalis strains. Interestingly, the Western blot results show thatmany of the NHA M. catarrhalis strains have OMP106 polypeptide in OGextracts of their outer membranes. This finding and the fact that silverstaining of OMPs from OG outer membrane extracts of NHA M. catarrhalisstrains after PAGE does not reveal a band in the 180 kD to 230 kD rangeindicate that OMP106 polypeptide is expressed by most M. catarrhalisstrains or cultivars but that, in order to be active in hemagglutination(i.e., binding to receptor on mammalian cell surfaces) or silverstainable, the OMP106 polypeptide must be appropriately modified in somemanner. Apparently only HA strains and cultivars are capable ofappropriately modifying OMP106 polypeptide so that it can mediatebacterial binding to hemagglutinin receptor on mammalian cell surfaces.

7. Example Efficacy of OMP106 Vaccine Cytotoxic Activity of Anti-OMP106Antiserum

Complement-mediated cytotoxic activity of anti-OMP106 antibodies wasexamined to determine the vaccine potential of OMP106 polypeptide.Antiserum to OMP106 polypeptide of a HA cultivar of ATCC® (American TypeCulture Collection) 49143 was prepared as described in Section 6.1.8.supra. The activities of the pre-immune serum and the anti-OMP106antiserum in mediating complement killing of M. catarrhalis wereexamined using the “Serum Bactericidal Test” described by Zollinger etal. (Immune Responses to Neisseria meningitis, in Manual of ClinicalLaboratory Immunology, 3rd ed., pg 347-349), except that cells of HA andNHA M. catarrhalis strains or cultivars were used instead of Neisseriameningitis cells.

The results show that anti-OMP106 antiserum mediated complement-killingof a HA cultivar of heterologous M. catarrhalis ATCC® (American TypeCulture Collection) 43627 but not a NHA cultivar of M. catarrhalis ATCC®(American Type Culture Collection) 43627 or the NHA M. catarrhalis ATCC®(American Type Culture Collection) 8176. Table 3 summarizes thecomplement mediated cytotoxic activities of pre-immune serum andanti-OMP106 antiserum against a HA cultivar of ATCC® (American TypeCulture Collection) 43627.

TABLE 3 Complement mediated cytotoxic activities of pre-immune serum andanti-OMP106 antiserum Cytotoxic Titer¹ Pre-immune Anti-OMP106 Experiment1 16 128 Experiment 2 8 64 ¹The titer is in the highest dilution atwhich a serum can mediate complement killing of a HA cultivar of ATCC ®(American Type Culture Collection) 43627 (e.g., 16 represents a 16 folddilution of the serum), the larger the number, the higher the cytotoxicactivity or titer.

As shown in Table 3, the anti-OMP106 antiserum has 8 fold greatercytotoxic activity than the pre-immune serum. This finding indicatesthat OMP106 polypeptide is useful as a vaccine against HA M. catarrhalisstrains and cultivars.

8. Example Isolation of the omp 106 Gene 8.1. Preparation of 72 bpPrimer

Degenerate PCR primers were designed based on the OMP106 N-terminalsequence information, including the 40 amino acid sequence depicted inSEQ ID NO:11.

A subset of this sequence (shown below) comprising 24 amino acids (SEQID NO:12) was chosen for the design of the degenerate oligonucleotidesMC 11 (SEQ ID NO:13) and MC 12 (SEQ ID NO:14), the sequences of whichare also shown below:

(SEQ ID NO: 12) EADGGKGGANARGDKSIAIGDIAQ MC 11: (SEQ ID NO: 13) GAR GCNGAY GGN GGN AAR (512-fold degenerate) MC 12: (SEQ ID NO: 14) YTG NGC DATRTC NCC DAT (576-fold degenerate).

These oligonucleotide primers were chosen to amplify an approximate 72bp DNA fragment of Moraxella catarrhalis omp 106 gene from genomic DNA.The size of the fragment was chosen to facilitate isolation byconventional agarose DNA electrophoresis. There is comparable degeneracyamong the primers, and the primers exclude sequences encoding serine,arginine or leucine.

8.2. PCR Amplification of a 72 bp DNA Fragment from Moraxellacatarrhalis Genomic DNA

A 72 bp DNA fragment was amplified from 20 ng of Moraxella catarrhalisgenomic DNA template using the degenerate oligonucleotides MC 11 (SEQ IDNO:13) and MC 12 (SEQ ID NO:14) (0.5 μM each) and 5 U of Taq polymeraseusing the following PCR program:

Hold 1: at 94° C. for 2 min. Cycle (x 30): Denature at 94° C. for 10 secAnneal at 45° C. for 15 sec Elongate at 72° C. for 4 sec Hold 2: at 72°C. for 1 min.

8.3. Isolation and Subcloning of the 72 bp PCR Amplification Product

The material from a 50 μl amplification assay was subjected toelectrophoresis on a 2% TAE agarose gel and the 72 bp DNA fragment wasexcised and recovered from the solubilized gel using a QiaQuick column(Qiagen). The isolated 72 bp DNA fragment was cloned into the pCR-ScriptAmpSK(+) vector (Stratagene) following the kit manufacturer's protocol.A miniprep DNA was prepared using standard protocols. Insert bearingplasmids were identified by restriction digestion with Pvu II, whichcuts on both sides of the plasmid polylinker. Plasmids containing the 72bp insert exhibited a proportionate size increase when compared to the448 bp Pvu II restriction fragment obtained from nonrecombinantplasmids.

8.4. Sequencing of the 72 bp Insert in PCR Script AMP SK(+)

The preparation of DNA templates and all sequencing protocols were doneas described by the manufacturer of the sequencing kit (USB). Thesequencing reactions were run on a 6% Acrylamide-7 M urea-TBE gel,fixed, dried and exposed to Kodak X-OMAT AR 5 film at room temperature.The sequence determined from these experiments is shown below alignedwith the encoded string of amino acids:

(SEQ ID NO: 18) Glu Ala Asp Gly Gly Lys Gly Gly Ala Asn Ala Arg Gly AspLys GAA GCG GAC GGG GGG AAA GGC GGA GCC AAT GCG CGC GGT GAT AAA Ser IleAla Ile Gly Asp Ile Ala Gln (SEQ ID NO: 17) TCC ATT GCT ATT GGT GAG ATTGCG CAA.

Thus, the amino acid sequence encoded by the cloned 72 bp DNA insertexactly matched the sequence obtained from the mature N-terminus of theOMP106 protein beginning at residue 6 of SEQ ID NO:11.

8.5. Generation of a Radiolabeled 72 bp DNA Screening Probe

The sequence information shown above was used to design a pair ofnondegenerate oligonucleotide primers, MC 17 (SEQ ID NO:15) AND MC 18(SEQ ID NO:16), respectively, whose sequences are as follows:

MC 17: GAA GCG GAC GGG GGG AAA (SEQ ID NO: 15) MC 18: TTG CGC AAT GTCACC AAT (SEQ ID NO: 16)

PCR amplification of the 72 bp DNA fragment was performed under the sameconditions as described above with the exception that the annealingtemperature was raised to 50° C. The 72 bp DNA fragment was isolatedfrom an agarose gel as before and radiolabeled using [32P]-γ-ATP and T4polynucleotide kinase according to standard methods. Unincorporatedradiolabel was separated from the probe on a G25 Sepharose spin column.Before use the probe was denatured for 2 min. at 95° C. and subsequentlychilled on ice (4° C.).

8.6. Hybridization of Plaque-Lift Filters and Southern Blots withRadiolabeled Probe

Phage plaques from library platings were immobilized on nylon filtersusing established transfer protocols. Digested bacterial genomic DNA,phage or plasmid DNA was electrophoresed on 0.8% TAE-agarose gels andtransferred onto nylon filters using a pressure blotter (Stratagene)according to the manufacturer's recommendations. Hybridizations with the72 bp probe were performed at 50° C. Hybridizations with other probeswere generally carried out at 60° C. Washes of increasing stringencywere done at the respective hybridization temperatures until nonspecificbackground was minimized.

8.7. Construction of a Moraxella catarrhalis Genomic DNA Library

A genomic library was constructed in the FIX II replacement vectorobtained from Stratgene. The vector arms were digested with Xho I andpartially filled-in by DNA polymerase (Klenow fragment) with dTTP anddCTP, resulting in 5′ TC overhangs. Partial digests of Moraxellacatarrhalis DNA by Sau 3A were performed to yield fragment sizes between9 kb and 23 kb. The cleaved DNA was partially filled-in by DNApolymerase with dGTP and dATP, resulting in 5′ GA overhangs. It will beobvious to those skilled in the art that neither partially filled-invector arms nor inserts will be able to self-ligate whereas thepartially filled-in vector and the partially filled-in inserts willligate. Ligations of vector arms and insert DNA were carried outaccording to standard protocols. Ligation reactions were packaged invitro using the Stratagene GigaPack Gold III extract. The packaged phagewere plated on E. coli X1 blue MRA (P2) (Stratagene) to further suppressnonrecombinants. The initial library titer was determined to beapproximately 2×10⁵ pfu.

The library was screened using 4×10⁴ pfu that were plated at a densityof 8×10³ pfu/130 mm plate. Several putative positive phage plaques werelocated and the six strongest hybridizing phage, omp106.1 to omp106.6,respectively, were eluted from cored agarose plugs, titered and replatedfor secondary screening. After the second screening only, omp106.3,omp106.5, and omp106.6, respectively, turned out to be true positives.These phage were replated at low density (approximately 100 pfu/plate)and three plaques per plate were analyzed by PCR using the primer pairMC17 (SEQ ID NO:15) and MC18 (SEQ ID NO:16) as described. The DNAamplification products from each of the three phage exactly matched thesize of the 72 bp PCR fragment obtained from genomic Moraxellacatarrhalis DNA.

8.8. Large Scale Phage DNA Isolation

One liter cultures of bacteria infected with individual phage eluateswere grown for about 6 hrs at which time lysis occurred. The lysateswere processed according to standard procedures. Crude phagepreparations were purified by banding in CsCl, digested with ProteinaseK, extracted with a 1:1 (v/v) mixture of phenol and chloroform andprecipitated with 2 volumes of ethanol after adjusting the DNA solutionto 0.3 M NaOAc (pH 7.8) all according to protocols well known to thoseskilled in the art.

8.9. Determination of Insert Size and Mapping of DNA FragmentsHybridizing with the 72 bp Probe

In order to estimate the size of the inserts in omp106.3, omp106.5, andomp106.6, phage DNA was digested with NotI and the digests were analyzedon a 0.5% TAE-agarose gel side by side with suitable DNA markers. Theapproximate sizes of the inserts were 11 kb, 15 kb and 20 kb for omp106.3, omp 106.5, and omp106.6, respectively. In order to maprestriction fragments that would hybridize to the 72 bp probe, DNA fromeach phage isolate was digested with a number of common restrictionenzymes either alone or in combination with NotI. The rationale of thisapproach was to discriminate between fragments that span theinsert/phage arm junction and those that map on the NotI insert. Theseries of single and double digests were run side-by-side for each phageisolate and analyzed by Southern analysis with radiolabeled 72 bp probe.These mapping experiments showed that the NotI insert of phage omp106.6contained the largest contiguous hybridizing restriction fragments ofall phage. Two of these fragments, a NotI/PstI fragment 5.5 kb and aHindIII fragment having an approximate size of 12 kb, respectively, werechosen for further analysis because they appeared to be large enough toharbor either the entire coding region for OMP106 or the majoritythereof.

8.10. Mapping of the 72 bp DNA region in the Subcloned Noti/PstiFragment of omp106.6

The NotI/PstI fragment was excised from omp106.6 phage DNA, isolatedfrom a 0.8% TAE-agarose gel and subcloned into the polylinker sites ofpBluescript II SK to yield the recombinant plasmid p omp N/P. Bacteriaharboring this plasmid grew unusually slowly and tended to delete andrearrange part of the insert, even more so when grown for large scaleplasmid DNA preps. Since this region contained sequences coding for theamino terminus of OMP106, it was likely that it would also carryregulatory upstream elements that were potentially active and/ordetrimental to the maintenance of the plasmid in E. coli. Likewise,leaky transcription from the plasmid lacZ promoter could have led toexpression of Moraxella sequences that were toxic for E. coli. A coarserestriction mapping was performed using enzymes that cut in the plasmidpolylinker. This analysis showed that the insert had a ClaI restrictionsite approximately 1.5 kb upstream from the PstI site. Likewise, atleast two HindIII restriction sites mapped approximately 2.8 kb andapproximately 3.5 kb upstream from the PstI site.

Next, the location of the 72 bp DNA fragment within the 5.5 kb NotI/PstIfragment was elucidated using a PCR-based mapping approach employing theScreenTest Screening Kit (Stratagene). This kit enables the user todetermine the orientation of an insert by amplifying the DNA sequencesbetween an insert-resident primer site and one that lies outside theplasmid's multiple cloning site.

The kit oligonucleotide P1 (TCATCATTGGAAAACGTTCTTCGGGGCGAA) SEQ ID NO:19hybridizes approximately 1 kb away from the multiple cloning site of pomp N/P. The size of a PCR product obtained with the oligonucleotides P1and MC 17 (SEQ ID NO:15) was approximately 1.5 kb. It was hence deducedthat the 72 bp fragment maps at approximately 450 bp upstream from thePstI site. It was also evident from this information that the major partof the OMP106 protein was encoded by sequences located beyond the PstIsite. By the same token, there was ample sequence upstream from the 72bp region to encode a presumptive signal sequence andpromoter/regulatory elements to drive transcription of this gene inMoraxella catarrhalis.

8.11. Mapping of the 12 Kb HindIII Insert from OMP106.6

The 12 kb HindIII fragment of omp106.6 was excised from phage DNA,separated on a 0.8% TAE-agarose gel and recovered as described above.The isolated DNA fragment was further digested with a number of commonrestriction endonucleases. Digestion with EcoRI cut the HindIII fragmentinto two 6 kb fragments, whereas digestion with PstI yielded restrictionfragments of approximately 3 kb and approximately 9 kb.

Based on the hybridization data with the 72 bp DNA probe, the mapping ofa HindIII site approximately 3 kb upstream of the PstI site in p omp N/Pand the mapping of a PstI site at a distance of approximately 3 kb fromone HindIII site in the 12 kb HindIII fragment, it was hypothesized thatthe 5.5 kb NotI/PstI fragment and the 12 kb HindIII fragment of phageomp106.6 might overlap in the 3 kb DNA sequence between HindIII andPstI. In view of the potential coding capacity of the 12 kb HindIIIfragment, it was concluded that this fragment would have enough sequenceinformation to encode the open reading frame of OMP106.

8.12. Subcloning of Restriction Fragments of omp106.6 and Analysis ofRecombinant Plasmids

The ClaI/PstI restriction fragment from p omp N/P was subcloned into therespective polylinker sites of pBluescript II SK to yield the plasmid pomp C/P. The mixture of HindIII/EcoRI fragments obtained by EcoRIdigestion of the 12 kb HindIII fragment was cloned into ZAP Express armsobtained by double digestion with EcoRI and HindIII. The phage/insertligations were packaged as described above and plated on a lawn of E.coli XL blue MRF′ cells (Stratagene). Recombinant phage were convertedinto insert bearing phagemids using the coinfection protocol withExAssist helper phage and the E. coli XLOLR strain provided with the kit(Stratagene). The resulting phagemids were screened for the presence ofEcoR/HindIII inserts of approximately 6 kb. Furthermore, diagnosticdigestions with PstI were performed to distinguish between clones havingthe promoter-proximal HindIII/EcoRI fragment and those carrying thepromoter-distal EcoRI/HindIII fragment. Invariably, in a large number ofanalyzed phagemids, the cloned insert did not cut with PstI. It wasconcluded that the insert having an approximate size of 6 kb was thepromoter-distal EcoRI/HindIII fragment of omp106.6. The resultingphagemid was designated as pBK omp R/H. Physical mapping of thisphagemid located a unique XbaI site approximately 1.5 kb upstream fromthe HindIII site. Sequences between this XbaI site and the XbaI site inthe polylinker were deleted and the recircularized phagemid wasdesignated as pBK omp R/X. On the basis of information described aboveherein it was calculated that the EcoRI/XbaI restriction fragment hadample sequence information to encode the C-terminal half of OMP106.

Finally, the missing sequences between the PstI site and the EcoRI weregenerated by long distance PCR (Barnes, W. M., 1994, Proc. Natl. Acad.Sci. USA 91:2216 2220) using genomic DNA or phageλ omp 106.6 DNA as thetemplate. The primers for this experiment were MC 17 (SEQ ID NO:15) anda gene-specific primer, omp R/X al (CGG TCA GCT TAG GCG TGG TT) (SEQ IDNO:20) which was designed based on sequence information downstream fromthe EcoRI site in pBK omp R/X. The PCR product having an approximatesize of 3.5 kb was digested with PstI and EcoRI and the approximately 3kb fragment was gel-isolated and cloned into PstIIEcoRI digestedpBluescript II SK. The resulting recombinant plasmid was designated as pomp P/R. A map of the omp106 locus, including fragments subcloned andused in various constructs, is shown in FIG. 11. Construction of theplasmids illustrated in FIG. 11 is described herein below in Section 9.

9. Example Sequencing of the omp 106 Gene

Sequencing of the omp 106 gene was performed using the plasmids p ompC/P, pBK omp R/X and p omp P/R as a template. Initially, commerciallyavailable T3 and T7 promoter primers flanking the multiple cloning sitein these plasmids were used as sequencing primers. Nested deletions ofthese plasmids were generated using the Exo Mung Bean Deletion kitpurchased from Stratagene following the manufacturer's protocol. Genespecific primers were designed on the basis of the sequence data asneeded.

Secondary confirmation of the sequences was obtained using a series ofgene-specific primers and omp106.6 phage DNA and PCR fragments amplifiedfrom Moraxella catarrhalis genomic DNA as the sequencing template. PCRfragments for this application were generated using the Advantagegenomic PCR kit from Clontech which contains a mix of highfidelity/proofreading polymerases. Sequencing from these templates wasdone to rule out potential cloning artifacts incurred during the geneticmanipulations. All sequencing reactions were performed using the DyeTerminator Cycle Sequencing Kit from Perkin-Elmer according to themanufacturer's specifications. The sequencing reactions were read usingan ABI Prism 310 Genetic Analyzer. The sequences were aligned using theAutoAssembler software (Perkin-Elmer) provided with the ABI Prism 310sequencer.

The EcoRI/XbaI fragment of omp 106 was released from pBK omp R/X as a 4kb NotI/BamHI fragment (by use of polylinker sites) and subcloned into pomp C/P that had been digested with the same enzymes. The p omp C/P/R/Xintermediate was then digested with PstI and EcoRI to receive the omp106 PstI/EcoRI fragment that had been isolated from p omp P/R. Thisconstruct, designated p omp 106X, contains the entire open reading frameof Moraxella catarrhalis OMP106 protein. The approximate size of theinsert is 8.5 kb. This plasmid was inserted into E. coli Top10(Invitrogen) and deposited with American Type Culture Collection (ATCC®)as E. coli Top10 (pOMP106).

The nucleotide sequence of the entire omp 106 gene is shown in SEQ IDNO:8. A deduced amino acid sequence of the open reading frame of omp 106is shown in SEQ ID NO:9.

10. Example Verification of the omp 106 Gene 10.1. Construction of anomp 106 Gene-Targeting Cassette

A gene targeting cassette was assembled from various omp 106 subclones(described above): p omp C/P, omp P/R, pBK omp R/X and a KanamycinResistance GenBlock purchased from Pharmacia. P omp 106X, was linearizedwith PstI and used to clone the Kanamycin resistance gene as a PstIfragment. Depending on the orientation of the kanamycin insert(determined by cutting an asymmetric ClaI site in the KanR gene block),the resulting constructs were designated as P omp X KO (the kanamycinand omp 106 genes are transcribed in the same direction) or p omp X OK(kanamycin and omp 106 gene transcription proceed towards each other).Highly purified DNA of both constructs was prepared using standardprotocols.

10.2. Preparation of Electrocompetent Moraxella Catarrhalis Cells

Moraxella catarrhalis cells were grown to an optical density (OD600 nm)of 1, harvested by centrifugation (3000×g), and subsequently washedtwice in ice-cold distilled water and once in 15% glycerol. The finalcell pellet was resuspended in 1 2 ml of 15% glycerol and rapidly frozenin 100 μl aliquots on dry ice. The electrocompetent cells were stored at−80° C.

10.3. Electroporation of Competent Cells

Aliquots (50 μl) of electrocompetent cells were mixed with 1 μg ofplasmid DNA, transferred to a 0.1 cm electroporation cuvette and kept onice for 1 min. An electroporation pulse was subsequently delivered usingthe following settings: 1500 V, 50 μF and 150′Ω. The pulsed culture wasimmediately transferred to Mueller-Hinton medium and incubated for 6 hrsat 37° C. Aliquots of the culture were then spread on selective mediaplates (Mueller-Hinton with 5 μg/ml of Kanamycin) and incubated at 37°C. until colonies were clearly visible (24-36 hrs). A random sample ofbacteria was picked and restreaked to obtain single colonies. Individualcolonies were grown in 2 ml cultures as above and used to preparegenomic DNA for PCR analysis.

11. Example Genetic Analysis 11.1. PCR Analysis

DNA from KAN^(R) Moraxella catarrhalis colonies was analyzed by PCRusing a forward primer that hybridizes upstream from the ClaIrestriction site (i.e. outside the sequences used in the targetingconstruct) and a reverse primer located in the coding region of thekanamycin gene. A PCR product is only to be expected if the incomingtargeting cassette has been integrated into the genome by homologousrecombination. No amplification product was obtained with wild-type DNA,whereas 100% of the DNA of KAN^(R) colonies yielded an amplificationproduct of the predicted size. The amplification products span the PstIrestriction site into which the kanamycin marker was cloned. Digestionof the amplification products with PstI yielded fragments of thepredicted size. A map of omp 106 and deletion mutants thereof is shownin FIG. 12.

11.2. Southern Analysis of omp 106

Genomic DNA from wild-type Moraxella catarrhalis and from PCR positivedeletion mutants was digested with ClaI and ClaI/EcoRI. The digests wereseparated on a 0.8% TAE-agarose gel and transferred to nylon membranesusing standard protocols. The blots were hybridized with ³²p labeledprobes prepared from either the omp 106 ClaI/PstI region or from theKanamycin resistance gene. Using the omp 106 ClaI/PstI probe, fragmentshaving approximate sizes of 9 kb and 4.6 kb were detected in the ClaIand ClaI/EcoRI digests, respectively, on DNA from all wild-type strainstested, whereas a unique DNA fragment having an approximate size of 1.8kb is detected in both digests on DNA from the deletion mutants. Thepresence of this unique, new restriction fragment demonstrates thesuccessful targeting of the omp 106 locus.

As expected, probing of the membrane with the kanamycin gene did notgenerate any signal in Moraxella catarrhalis wild-type DNA. In DNA fromthe deletion mutants, the kanamycin probe detected fragments havingapproximate sizes of 9 kb and 1.8 kb and 4 kb and 1.8 kb in the ClaI andClaT/EcoRI digests, respectively. As before, the presence of thesesequences in the deletion mutants and their absence in the wild-type DNAdemonstrate that the omp 106 locus has been successfully altered.

12. Example Generation and Reactivity of Monoclonal Anti-OMP106Antibodies

BALB/c mice were immunized with total outer membranes from M.catarrhalis. Hybridomas for monoclonal antibodies were prepared byfusing the spleen cells from these mice to SP2/0 cells and selecting forsuccessful hybrids with HAT containing media. Reactive hybridomas werescreened using an ELISA containing detergent extracts of the total outermember of M. catarrhalis MC2926. From this screen, 12 hybridomas withvarying levels of activity in the ELISA were selected for clonalselection, the monoclonal antibodies were assayed for reactivity topurified OMP106 and total outer membranes from M. catarrhalisMC2926ΔOMP106 by ELISA. Monoclonal antibodies designated SRB #1 and SRB#3 both reacted specifically to OMP106 in the ELISA.

Western blots were performed as described in Example 6.1.9, usingmonoclonal antibodies SRB #1 and #3. SRB #1 demonstrated reactivity toOMP106 in Western blots to both the nondenatured form of the protein(greater than 250 kDa) and to the denatured form of the protein(approximately 190 kDa) produced by incubating the sample at 100° C. for5 minutes prior to resolving on the polyacrylamide gel (FIG. 10), whichsuggests that SRB #1 is reacting with a linear epitope from OMP106. SRB#3 did not react with OMP106 in the Western, regardless of the form ofthe protein, but as noted above, did react in an ELISA. This indicatesthat SRB #3 reacts with a native conformation of OMP106 that is notpresented in the Western blots.

13. Example Binding and Inhibition of Binding to Nasopharyngeal Cells13.1. Nasopharyngeal Cell Binding

The binding of Moraxella to the continuous cell line Hep-2 was assayedusing a modification of the procedure described by Galan and Curtiss (J.E. Galan and R. Curtiss III. 1989, Proc. Natl. Acad. Sci. USA86:6383-6387, incorporated herein by reference in its entirety). The M.catarrhalis strain MC2926 and MC2926ΔOMP106 were used to assay thebinding of Moraxella to Hep-2 cells. The MC2926ΔOMP106 strain is anisogenic strain to MC2926 but with the gene for OMP106 disrupted (asdescribed in Example 8 above), thereby causing the loss of theexpression of the OMP106 protein.

Briefly, the strains were grown to mid-log phase in Mueller Hintonbroth. Bacterial cells from the culture were then centrifuged onto themonolayer of Hep-2 cells and allowed to bind to the cells for 1 hour.Nonbound cells were removed by washing with Hanks balanced salt solutioncontaining calcium. Adherent cells were removed with the monolayer bytreatment with 0.1% sodium glycocholate in phosphate buffered saline(PBS). The number of adherent cells were enumerated by plating onMueller Hinton agar and allowing the bacteria to grow for 24 hours. Theefficiency of binding of the bacteria is expressed as a percentage ofbacteria bound relative to the original number of bacteria added to theHep-2 monolayer, and is shown in Table 4 below.

TABLE 4 Binding efficiency of MC2926 and the genetic deletion of omp 106(MC2926ΔOMP106) to HEp-2 cells. Bacterial strain % bound MC2926 100%MC2926ΔOMP106  21%

The results of the nasopharyngeal cell binding assay shows that OMP106is responsible for binding and adherence of Moraxella catarrhalis tonasopharyngeal cells.

13.2. Inhibition of Nasopharyngeal Binding

The hybridoma culture supernatants from Example 9 were used to treatbacterial cells to assay for their ability to block binding of theMoraxella bacteria to nasopharyngeal cells (HEp-2) by incubating thebacteria with monoclonal antibody for 5 minutes prior to exposing thebacteria to the HEp-2 cells. The data from this experiment indicatesthat one of the monoclonals (SRB #3) blocked the bacteria from bindingto nasopharyngeal cells (Table 5). This data, when considered with thedata from the strain with the genetic deletion of OMP106 (MC2926ΔOMP106)(Table 4), supports the conclusion that SRB #3 binds to a nativeconformation of OMP106. Furthermore, this demonstrates that SRB #3 canneutralize the biological activity of OMP106 (i.e., binding toeukaryotic cells). The observation that only one hybridoma blocked thebinding to nasopharyngeal cells, while the other did not rules out thepossibility that media components are responsible for the inhibition ofbinding.

TABLE 5 Antibody mediated inhibition of MC2926 binding to Hep-2 cellsTreatment of bacterial cells % bound Tissue culture media 98% HybridomaSRB #1 100% Hybridoma SRB #3 5%

14. Example Hemagglutination Assay

The hemagglutination assay described in Example 6.1 was performed usingthe wild-type and deletion mutant strains of M. catarrhalis. Thewild-type strain, MC2926, strongly agglutinates human red blood cells,whereas the isogenic mutant, MC2926ΔOMP106, does not hemagglutinate.This demonstrates that OMP106 is a hemagglutinin from M. catarrhalis.

15. Deposit of Microorganism

E. coli Top10 containing plasmid OMP106X (pOMP106X), was deposited onNov. 6, 1997 with the American Type Culture Collection (ATCC®), 1201Parklawn Drive, Rockville, Md. 20852, under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedures, and assignedaccession No. 98579.

The present invention is not to be limited in scope by the microorganismdeposited or the specific embodiments described herein. It will beunderstood that variations which are functionally equivalent are withinthe scope of this invention. Indeed, various modifications of theinvention, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of producing an immune response in an animal comprisingimmunizing the animal with an effective amount of an isolated orsubstantially pure OMP106 polypeptide, which is an outer membranepolypeptide of Moraxella catarrhalis, and which has a molecular weightof about 180 kD to about 230 kD as determined in SDS polyacrylamide gelelectrophoresis using rabbit skeletal muscle myosin and E. coliβ-galactosidase as the 200 kD and 116.25 kD molecular weight standards,respectively, and which comprises the amino acid sequence of SEQ IDNO:
 1. 2. The method according to claim 1 in which the OMP106polypeptide has a molecular weight of about 190 kD.
 3. The methodaccording to claim 1 in which the Moraxella catarrhalis is ahemagglutinating cultivar.
 4. The method according to claim 1 in whichthe effective amount of the isolated or substantially pure polypeptideis between 0.1 and 500 micrograms per dose.
 5. The method according toclaim 1 in which the isolated or substantially pure polypeptide isadministered with a pharmaceutically acceptable carrier.
 6. The methodaccording to claim 5 in which the pharmaceutically acceptable carrier isa stabilizer, a diluent, a buffer or combinations thereof.
 7. The methodaccording to claim 6 in which the isolated or substantially purepolypeptide is administered with an adjuvant.
 8. The method according toclaim 7 in which the adjuvant is a peptide, aluminum hydroxide, aluminumphosphate, aluminum oxide or combinations thereof.
 9. A method ofproducing an immune response in an animal comprising immunizing theanimal with an immunogenic composition comprising an isolated orsubstantially pure OMP106 polypeptide, which is an outer membranepolypeptide of Moraxella catarrhalis, and which has a molecular weightof about 180 kD to about 230 kD as determined in SDS polyacrylamide gelelectrophoresis using rabbit skeletal muscle myosin and E. coliβ-galactosidase as the 200 kD and 116.25 kD molecular weight standards,respectively, and which comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:
 9. 10. The method according to claim 9 in which thecomposition further comprises a pharmaceutically acceptable carrier. 11.The method according to claim 10 in which the pharmaceuticallyacceptable carrier is a stabilizer, a diluent, a buffer or combinationsthereof.
 12. The method according to claim 11 in which the stabilizercomprises carbohydrates, proteins, or both.
 13. The method according toclaim 12 in which the carbohydrate comprises sorbitol, lactose, manitol,starch, sucrose, dextran, glucose or combinations thereof.
 14. Themethod according to claim 12 in which the protein comprises albumin,casein or both.
 15. The method according to claim 11 in which thediluent comprises saline, Hanks Balanced Salts, Ringers solution orcombinations thereof.
 16. The method according to claim 11 in which thebuffer comprises an alkali metal phosphate, an alkali metal carbonate,an alkaline earth metal carbonate or combinations thereof.
 17. Themethod according to claim 9 in which the composition further comprisesan adjuvant.
 18. The method according to claim 17 in which the adjuvantcomprises a peptide, aluminum hydroxide, aluminum phosphate, aluminumoxide, mineral oil, a vegetable oil, a emulsifying agent, a surfaceactive substance, BCG, Corynebacterium parvum, or combinations thereof.19. The method according to claim 18 in which the surface activesubstance comprises lysolecithin, polycations, polyanions, orcombinations thereof.
 20. The method according to claim 9 in which thecomposition further comprises a second immunogen.